Notch1 receptor binding agents and methods of use thereof

ABSTRACT

The present invention relates to compositions and methods for characterizing, diagnosing, and treating cancer. In particular the invention provides the means and methods for the diagnosis, characterization, prognosis and treatment of cancer and specifically targeting cancer stem cells. The present invention provides an antibody that specifically binds to a non-ligand binding membrane proximal region of the extracellular domain of a human Notch receptor and inhibits tumor growth. The present invention further provides a method of treating cancer, the method comprising administering a therapeutically effective amount of an antibody that specifically binds to a non-ligand binding membrane proximal region of the extracellular domain of a human Notch receptor protein and inhibits tumor growth.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 13/003,013,filed Jul. 8, 2009, which is the U.S. national stage application ofInternational Application No. PCT/US2009/003995, filed Jul. 8, 2009,which claims the priority benefit of U.S. Provisional Application No.61/112,699, filed Nov. 7, 2008, U.S. Provisional Application No.61/112,701, filed Nov. 7, 2008, and U.S. Provisional Application No.61/079,095, filed Jul. 8, 2008, each of which is hereby incorporated byreference herein in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name:SequenceListing.txt; Size: 43 kilobytes; and Date of Creation: Mar. 13,2013) filed herewith is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions comprising an agent thatbinds a human Notch receptor and methods of using the compositions forcharacterizing, diagnosing, and treating cancer and other diseases. Inparticular, the present invention provides antibodies that specificallybind to a non-ligand binding membrane proximal region of theextracellular domain of a human Notch1 receptor and inhibit tumorgrowth. The present invention further provides methods of treatingcancer, the methods comprising administering a therapeutically effectiveamount of an antibody that specifically binds to a non-ligand bindingmembrane proximal region of the extracellular domain of a human Notch1receptor protein and inhibits tumor growth.

2. Background

Cancer is one of the leading causes of death in the developed world,resulting in over 500,000 deaths per year in the United States alone.Over one million people are diagnosed with cancer in the U.S. each year,and overall it is estimated that more than 1 in 3 people will developsome form of cancer during their lifetime. Though there are more than200 different types of cancer, four of them—breast, lung, colorectal,and prostate—account for over half of all new cases (Jemal et al. 2003,Cancer J. Clin. 53:5-26).

Cancer arises from dysregulation of the mechanisms that control normaltissue development and maintenance, and increasingly stem cells arethought to play a central role (Beachy et al., 2004, Nature 432:324).During normal animal development, cells of most or all tissues arederived from normal precursors, called stem cells (Morrison et al.,1997, Cell 88:287-98; Morrison et al., 1997, Curr. Opin. Immunol.9:216-21; Morrison et al., 1995, Annu. Rev. Cell. Dev. Biol. 11:35-71).Stem cells are cells that: (1) have extensive proliferative capacity; 2)are capable of asymmetric cell division to generate one or more kinds ofprogeny with reduced proliferative and/or developmental potential; and(3) are capable of symmetric cell divisions for self-renewal orself-maintenance. The best-known example of adult cell renewal by thedifferentiation of stem cells is the hematopoietic system wheredevelopmentally immature precursors (hematopoietic stem and progenitorcells) respond to molecular signals to form the varied blood andlymphoid cell types. Other cells, including cells of the gut, breastductal system, and skin are constantly replenished from a smallpopulation of stem cells in each tissue, and recent studies suggest thatmost other adult tissues also harbor stem cells, including the brain.

Solid tumors are composed of heterogeneous cell populations. Forexample, breast cancers are a mixture of cancer cells and normal cells,including mesenchymal (stromal) cells, inflammatory cells, andendothelial cells. Classic models of cancer hold that phenotypicallydistinct cancer cell populations all have the capacity to proliferateand give rise to a new tumor. In the classical model, tumor cellheterogeneity results from environmental factors as well as ongoingmutations within cancer cells resulting in a diverse population oftumorigenic cells. This model rests on the idea that all populations oftumor cells would have some degree of tumorigenic potential. (Pandis etal., 1998, Genes, Chromosomes & Cancer 12:122-129; Kuukasjrvi et al.,1997, Cancer Res. 57:1597-1604; Bonsing et al., 1993, Cancer 71:382-391;Bonsing et al., 2000, Genes Chromosomes & Cancer 82: 173-183; Beerman Het al., 1991, Cytometry 12:147-54; Aubele M & Werner M, 1999, Analyt.Cell. Path. 19:53; Shen L et al., 2000, Cancer Res. 60:3884).

An alternative model for the observed solid tumor cell heterogeneity isthat solid tumors result from a “solid tumor stem cell” (or “cancer stemcell” from a solid tumor) that subsequently undergoes chaoticdevelopment through both symmetric and asymmetric rounds of celldivisions. In this stem cell model, solid tumors contain a distinct andlimited (possibly even rare) subset of cells that share the propertiesof normal “stem cells”, in that they extensively proliferate andefficiently give rise both to additional solid tumor stem cells(self-renewal) and to the majority of tumor cells of a solid tumor thatlack tumorigenic potential. Indeed, mutations within a long-lived stemcell population may initiate the formation of cancer stem cells thatunderlie the growth and maintenance of tumors and whose presencecontributes to the failure of current therapeutic approaches.

The stem cell nature of cancer was first revealed in the blood cancer,acute myeloid leukemia (AML) (Lapidot et al., 1994, Nature 367:645-8).More recently it has been demonstrated that malignant human breasttumors similarly harbor a small, distinct population of cancer stemcells enriched for the ability to form tumors in immunodeficient mice.An ESA+, CD44+, CD24−/low, Lin-cell population was found to be 50-foldenriched for tumorigenic cells compared to unfractionated tumor cells(Al-Hajj et al., 2003, PNAS 100:3983-8). The ability to prospectivelyisolate the tumorigenic cancer cells has permitted investigation ofcritical biological pathways that underlie tumorigenicity in thesecells, and thus promises the development of better diagnostic assays andtherapeutics for cancer patients. It is toward this purpose that thisinvention is directed.

Normal stem cells and cancer stem cells share the ability to proliferateand self-renew, thus it is not surprising that a number of genes thatregulate normal stem cell development contribute to tumorigenesis(reviewed in Reya et al., 2001, Nature 414:105-111 and Taipale & Beachy,2001, Nature 411:349-354). The present invention identifies Notchreceptor, for example, Notch1, as a marker of cancer stem cells,implicating the Notch signaling pathway in the maintenance of cancerstem cells and as a target for treating cancer via the elimination ofthese tumorigenic cells.

The Notch signaling pathway is one of several critical regulators ofembryonic pattern formation, post-embryonic tissue maintenance, and stemcell biology. More specifically, Notch signaling is involved in theprocess of lateral inhibition between adjacent cell fates and plays animportant role in cell fate determination during asymmetric celldivisions. Unregulated Notch signaling is associated with numerous humancancers where it can alter the developmental fate of tumor cells tomaintain them in an undifferentiated and proliferative state (Brennanand Brown, 2003, Breast Cancer Res. 5:69). Thus carcinogenesis canproceed by usurping homeostatic mechanisms controlling normaldevelopment and tissue repair by stem cell populations (Beachy et al.,2004, Nature 432:324).

The Notch receptor was first identified in Drosophila mutants withhaploinsufficiency resulting in notches at the wing margin, whereasloss-of-function produces an embryonic lethal “neurogenic” phenotypewhere cells of the epidermis switch fate to neural tissue (Moohr, 1919,Genet. 4:252; Poulson, 1937, PNAS 23:133; Poulson, 1940, J. Exp. Zool.83:271). The Notch receptor is a single-pass transmembrane receptorcontaining numerous tandem epidermal growth factor (EGF)-like repeatsand three cysteine-rich Notch/LIN-12 repeats (LNRs) within a largeextracellular domain (Wharton et al., 1985, Cell 43:567; Kidd et al.,1986, Mol. Cell. Biol. 6:3094; reviewed in Artavanis et al., 1999,Science 284:770). The LNRs and an additional C-terminal tail ofapproximately 103 amino acids of the extracellular domain are referredto herein as the “membrane proximal region”. This region is also knownas, and referred to as the Notch negative regulatory region (NRR).

Mammalian Notch receptors undergo cleavage to both form the maturereceptor and following ligand binding to activate downstream signaling.A furin-like protease cleaves the Notch receptor precursors duringmaturation to generate juxtamembrane heterodimers that comprise anon-covalently associated extracellular subunit and a transmembranesubunit held together in an auto-inhibitory state. Ligand bindingrelieves this inhibition and induces cleavage of the Notch receptor byan ADAM-type metalloprotease and gamma-secretase, the latter of whichreleases the intracellular domain (ICD) into the cytoplasm, allowing itto translocate into the nucleus to activate gene transcription. Cleavageby ADAM occurs within the non-ligand binding cleavage domain within thejuxtamembrane negative regulatory region (NRR) (See FIG. 1A). In theNotch1 receptor this region encompasses from about amino acid 1427 toabout amino acid 1732.

Four mammalian Notch proteins have been identified (Notch1, Notch2,Notch3, and Notch4), and mutations in these receptors invariably resultin developmental abnormalities and human pathologies including severalcancers as described in detail below (Gridley, 1997, Mol. Cell.Neurosci. 9:103; Joutel & Tournier-Lasserve, 1998, Semin. Cell Dev.Biol. 9:619-25).

The Notch receptor is activated by single-pass transmembrane ligands ofthe Delta, Serrated, Lag-2 (DSL) family. There are five known Notchligands in mammals: Delta-like 1 (DLL1), Delta-like 3 (DLL3), Delta-like4 (DLL4), Jagged 1 and Jagged 2 characterized by a DSL domain and tandemEGF-like repeats within the extracellular domain. The extracellulardomain of the Notch receptor interacts with that of its ligands,typically on adjacent cells, resulting in two proteolytic cleavages ofNotch; one extracellular cleavage mediated by an ADAM (A Disintegrin AndMetallopeptidase) protease and one cleavage within the transmembranedomain mediated by gamma secretase. This latter cleavage generates theNotch intracellular domain (ICD), which then enters the nucleus where itactivates the CBF 1, Suppressor of Hairless [Su(H)], Lag-2 (CSL) familyof transcription factors as the major downstream effectors to increasetranscription of nuclear basic helix-loop-helix transcription factors ofthe Hairy and Enhancer of Split [E(spl)] family (Artavanis et al., 1999,Science 284:770; Brennan and Brown, 2003, Breast Cancer Res. 5:69; Isoet al., 2003, Arterioscler. Thromb. Vasc. Biol. 23:543). Alternativeintracellular pathways involving the cytoplasmic protein Deltexidentified in Drosophila may also exist in mammals (Martinez et al.,2002, Curr. Opin. Genet. Dev. 12:524-33), and this Deltex-dependentpathway may act to suppress expression of Wnt target genes (Brennan etal., 1999, Curr. Biol. 9:707-710; Lawrence et al., 2001, Curr. Biol.11:375-85).

Hematopoietic stem cells (HSCs) are the best understood stem cells inthe body, and Notch signaling is implicated both in their normalmaintenance as well as in leukemic transformation (Kopper & Hajdu, 2004,Pathol. Oncol. Res. 10:69-73). HSCs are a rare population of cells thatreside in a stromal niche within the adult bone marrow. These cells arecharacterized both by a unique gene expression profile as well as anability to continuously give rise to more differentiated progenitorcells to reconstitute the entire hematopoietic system. Constitutiveactivation of Notch1 signaling in HSCs and progenitor cells establishesimmortalized cell lines that generate both lymphoid and myeloid cells invitro and in long-term reconstitution assays (Varnum-Finney et al.,2000, Nat. Med. 6:1278-81), and the presence of Jagged1 increasesengraftment of human bone marrow cell populations enriched for HSCs(Karanu et al., 2000, J. Exp. Med. 192:1365-72). More recently, Notchsignaling has been demonstrated in HSCs in vivo and shown to be involvedin inhibiting HSC differentiation. Furthermore, Notch signaling appearsto be required for Wnt-mediated HSC self-renewal (Duncan et al., 2005,Nat. Immunol. 6:314).

The Notch signaling pathway also plays a central role in the maintenanceof neural stem cells and is implicated both in their normal maintenanceas well as in brain cancers (Kopper & Hajdu, 2004, Pathol. Oncol. Res.10:69-73; Purow et al., 2005, Cancer Res. 65:2353-63; Hallahan et al.,2004, Cancer Res. 64:7794-800). Neural stem cells give rise to allneuronal and glial cells in the mammalian nervous system duringdevelopment, and more recently have been identified in the adult brain(Gage, 2000, Science 287:1433-8). Mice deficient for Notch1; the Notchtarget genes Hes1, 3, and 5; and a regulator of Notch signalingpresenilin1 (PS1) show decreased numbers of embryonic neural stem cells.Furthermore, adult neural stem cells are reduced in the brains of PS1heterozygote mice (Nakamura et al., 2000, J. Neurosci. 20:283-93;Hitoshi et al., 2002, Genes Dev. 16:846-58). The reduction in neuralstem cells appears to result from their premature differentiation intoneurons (Hatakeyama et al., 2004, Dev. 131:5539-50) suggesting thatNotch signaling regulates neural stem cell differentiation andself-renewal.

Aberrant Notch signaling is implicated in a number of human cancers. TheNotch1 gene in humans was first identified in a subset of T-cell acutelymphoblastic leukemias as a translocated locus resulting in activationof the Notch pathway (Ellisen et al., 1991, Cell 66:649-61).Constitutive activation of Notch1 signaling in T-cells in mouse modelssimilarly generates T-cell lymphomas suggesting a causative role (Robeyet al., 1996, Cell 87:483-92; Pear et al., 1996, J. Exp. Med.183:2283-91; Yan et al., 2001, Blood 98:3793-9; Bellavia et al., 2000,EMBO J. 19:3337-48). Recently Notch1 point mutations, insertions, anddeletions producing aberrant Notch1 signaling have been found to befrequently present in both childhood and adult T-cell acutelymphoblastic leukemia/lymphoma (Pear & Aster, 2004, Curr. Opin.Hematol. 11:416-33).

The frequent insertion of the mouse mammary tumor virus into both theNotch1 and Notch4 locus in mammary tumors and the resulting activatedNotch protein fragments first implicated Notch signaling in breastcancer (Gallahan & Callahan, 1987, J. Virol. 61:66-74; Brennan & Brown,2003, Breast Cancer Res. 5:69; Politi et al., 2004, Semin. Cancer Biol.14:341-7). Further studies in transgenic mice have confirmed a role forNotch in ductal branching during normal mammary gland development, and aconstitutively active form of Notch4 in mammary epithelial cellsinhibits epithelial differentiation and results in tumorigenesis(Jhappan et al., 1992, Genes & Dev. 6:345-5; Gallahan et al., 1996,Cancer Res. 56:1775-85; Smith et al., 1995, Cell Growth Differ.6:563-77; Soriano et al., 2000, Int. J. Cancer 86:652-9; Uyttendaele etal., 1998, Dev. Biol. 196:204-17; Politi et al., 2004, Semin. CancerBiol. 14:341-7). Currently the evidence for a role for Notch in humanbreast cancer is limited to the expression of Notch receptors in breastcarcinomas and their correlation with clinical outcome (Weijzen et al.,2002, Nat. Med. 8:979-86; Parr et al., 2004, Int. J. Mol. Med.14:779-86). Furthermore, overexpression of the Notch pathway has beenobserved in cervical cancers (Zagouras et al., 1995, PNAS 92:6414-8),renal cell carcinomas (Rae et al., 2000, Int. J. Cancer 88:726-32), headand neck squamous cell carcinomas (Leethanakul et al., 2000, Oncogene19:3220-4), endometrial cancers (Suzuki et al., 2000, Int. J. Oncol.17:1131-9), and neuroblastomas (van Limpt et al., 2000, Med. Pediatr.Oncol. 35:554-8) suggestive of a potential role for Notch in thedevelopment of a number of neoplasms. Interestingly, Notch signalingmight play a role in the maintenance of the undifferentiated state ofApc-mutant neoplastic cells of the colon (van Es & Clevers, 2005, Trendsin Mol. Med. 11:496-502).

The Notch pathway is also involved in multiple aspects of vasculardevelopment including proliferation, migration, smooth muscledifferentiation, angiogenesis and arterial-venous differentiation (Isoet al., 2003, Arterioscler. Thromb. Vasc. Biol. 23:543). For example,homozygous null mutations in Notch1/4 and Jagged1 as well asheterozygous loss of DLL4 result in severe though variable defects inarterial development and yolk sac vascularization. Furthermore,DLL1-deficient and Notch-2-hypomorphic mice embryos show hemorrhage thatlikely results from poor development of vascular structures (Gale etal., 2004, PNAS, 101:15949-54; Krebs et al., 2000, Genes Dev.14:1343-52; Xue et al., 1999, Hum. MeI Genet. 8:723-30; Hrabe de Angeliset al., 1997, Nature 386:717-21; McCright et al., 2001, Dev.128:491-502). In human, mutations in Jagged1 are associated withAlagille syndrome, a developmental disorder that includes vasculardefects, and mutations in Notch3 are responsible for an inheritedvascular dementia (Cadasil) in which vessel homeostasis is defective(Joutel et al., 1996, Nature 383:707-10).

The identification of Notch1, Notch4, DLL1 and DLL4 as genes expressedin cancer stem cells compared to normal breast epithelium suggests thattargeting the Notch pathway can help eliminate not only the majority ofnontumorigenic cancer cells, but the tumorigenic cells responsible forthe formation and reoccurrence of solid tumors. Furthermore, because ofthe prominent role of angiogenesis in tumor formation and maintenance,targeting the Notch pathway can also effectively inhibit angiogenesis,starving a cancer of nutrients and contributing to its elimination.

Anti-Notch antibodies and their possible use as anti-cancer therapeuticshave been reported. See, e.g., U.S. Patent Application Publication Nos.2008/0131434 and 2009/0081238, each of which is incorporated byreference herein in its entirety. See also International PublicationNos. WO 2008/057144, WO 2008/076960, and WO 2008/50525.

BRIEF SUMMARY OF THE INVENTION

The present invention provides agents that bind to a non-ligand bindingmembrane proximal region of the extracellular domain of a Notch1receptor and compositions, such as pharmaceutical compositions,comprising those agents. The invention further provides methods oftargeting cancer stem cells with the agents. In some embodiments, themethods comprise reducing the frequency of cancer stem cells in a tumor,reducing the number of cancer stem cells in a tumor, reducing thetumorigenicity of a tumor, and/or reducing the tumorigenicity of a tumorby reducing the number or frequency of cancer stem cells in the tumor.The invention also provides methods of using the agents in the treatmentof cancer and/or in the inhibition of tumor growth.

In one aspect, the invention provides an antibody that specificallybinds to a non-ligand binding membrane proximal region of theextracellular domain of a Notch1 receptor (e.g., human Notch1). In someembodiments, the non-ligand binding membrane proximal region of a Notch1receptor comprises about amino acid 1427 to about amino acid 1732 of ahuman Notch1 receptor. In some embodiments, the membrane proximal regionof a Notch1 receptor comprises SEQ ID NO:2. In certain embodiments, theantibody specifically binds to a non-ligand binding membrane proximalregion of the extracellular domain of at least one additional Notchreceptor family member.

In some embodiments the antibody is an antagonist of Notch1. In someembodiments, the antibody inhibits signaling by or activation of theNotch1 receptor. In some embodiments, the antibody inhibits Notch1activity. In some embodiments, the antibody inhibits cleavage within themembrane proximal region. In certain embodiments, the antibody inhibitscleavage of the Notch1 receptor (e.g., cleavage at the S2 site by ametalloprotease) and/or inhibits activation of the Notch1 receptor byligand binding. In some embodiments, the antibody inhibits release orformation of the intracellular domain (ICD) of Notch1. In certainembodiments, the antibody inhibits tumor growth.

In certain embodiments, the invention provides an antibody that binds anon-ligand binding membrane proximal region of the extracellular domainof a human Notch1 and comprises a heavy chain CDR1 comprising RGYWIE(SEQ ID NO:15), a heavy chain CDR2 comprising QILPGTGRTNYNEKFKG (SEQ IDNO:16), and/or a heavy chain CDR3 comprising FDGNYGYYAMDY (SEQ IDNO:17); and/or (b) a light chain CDR1 comprising RSSTGAVTTSNYAN (SEQ IDNO:18), a light chain CDR2 comprising GTNNRAP (SEQ ID NO:19), and/or alight chain CDR3 comprising ALWYSNHWVFGGGTKL (SEQ ID NO:20). In someembodiments, the antibody comprises a heavy chain variable regioncomprising: (a) a heavy chain CDR1 comprising RGYWIE (SEQ ID NO:15), ora variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (b)a heavy chain CDR2 comprising QILPGTGRTNYNEKFKG (SEQ ID NO:16), or avariant thereof comprising 1, 2, 3, or 4 amino acid substitutions;and/or (c) a heavy chain CDR3 comprising FDGNYGYYAMDY (SEQ ID NO:17), ora variant thereof comprising 1, 2, 3, or 4 amino acid substitutions. Incertain other embodiments, the antibody comprises (or further comprises)a light chain variable region comprising: (a) a light chain CDR1comprising RSSTGAVTTSNYAN (SEQ ID NO:18), or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions; (b) a light chainCDR2 comprising GTNNRAP (SEQ ID NO:19), or a variant thereof comprising1, 2, 3, or 4 amino acid substitutions; and/or (c) a light chain CDR3comprising ALWYSNHWVFGGGTKL (SEQ ID NO:20), or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions. In some embodiments,the amino acid substitutions are conservative amino acid substitutions.

In some embodiments, the invention provides an antibody, 52M51, producedby the hybridoma cell line deposited with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va., USA, underthe conditions of the Budapest Treaty on Aug. 7, 2008, and assigneddesignation number PTA-9405. In some embodiments, the invention providesa humanized version of antibody 52M51, 52M51H4L3, as encoded by the DNAdeposited with the ATCC, under the conditions of the Budapest Treaty onOct. 15, 2008, and assigned designation number PTA-9549. In someembodiments, the invention provides an antibody that binds to the sameepitope as the epitope to which antibody 52M51 binds.

In another aspect, the invention provides an antibody that binds anon-ligand binding membrane proximal region of the extracellular domainof a human Notch1 and the antibody comprises, consists, or consistsessentially of an antibody “52R43” as encoded by the DNA deposited withthe ATCC under the conditions of the Budapest Treaty on Oct. 15, 2008,and assigned designation number PTA-9548. In some embodiments, theinvention provides an antibody that competes with 52R43 for specificbinding to a non-ligand binding membrane proximal region of theextracellular domain of a human Notch1. Pharmaceutical compositionscomprising 52R43 and methods of treating cancer comprising administeringtherapeutically effective amounts of the 52R43 antibody are alsoprovided.

In certain embodiments, the invention provides an antibody that competeswith any of the antibodies as described in the aforementionedembodiments and/or aspects, as well as other aspects/embodimentsdescribed elsewhere herein, for specific binding to a non-ligand bindingmembrane proximal region of the extracellular domain of a human Notch1(e.g., in a competitive binding assay). Pharmaceutical compositionscomprising the antibodies described herein and methods of treatingcancer comprising administering therapeutically effective amounts of theantibodies are also provided.

In certain embodiments of each of the aforementioned aspects orembodiments, as well as other aspects and/or embodiments describedelsewhere herein, the antibody is a recombinant antibody. In someembodiments, the antibody is a monoclonal antibody, a chimeric antibody,a humanized antibody, or a human antibody. In certain embodiments, theantibody is an antibody fragment. In certain embodiments, the antibodyor antibody fragment is monovalent, monospecific, bivalent, bispecific,or multispecific. In certain embodiments, the antibody is conjugated toa cytotoxic moiety. In certain embodiments, the antibody is isolated. Instill further embodiments, the antibody is substantially pure.

Pharmaceutical compositions comprising the antibodies described hereinand methods of treating cancer comprising administering therapeuticallyeffective amounts of the antibodies described herein are also provided.In certain embodiments, the pharmaceutical compositions further comprisea pharmaceutically acceptable carrier.

In another aspect, the invention provides a polypeptide. In someembodiments, the polypeptide is an antibody (e.g., an antibody thatspecifically binds Notch1), a heavy chain or light chain of an antibody,and/or a fragment of an antibody. In some embodiments, the polypeptideis isolated. In certain embodiments, the polypeptide is substantiallypure. In some embodiments, the polypeptide comprises an amino acidsequence of SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:28, orSEQ ID NO:32. In some embodiments, the polypeptide comprises an aminoacid sequence of SEQ ID NO:14 or SEQ ID NO:24 and/or an amino acidsequence of SEQ ID NO:8, SEQ ID NO:28, or SEQ ID NO:32. In someembodiments, the polypeptide comprises at least a portion of the aminoacid sequence of SEQ ID NO:14 or SEQ ID NO:24, and/or at least a portionof the amino acid sequence of SEQ ID NO:8, SEQ ID NO:28, or SEQ IDNO:32. Pharmaceutical compositions comprising both the polypeptide and apharmaceutically acceptable vehicle are further provided, as are celllines that produce the polypeptide.

In some embodiments, the polypeptide comprises: (a) a polypeptide havingat least about 80% sequence identity to SEQ ID NO:14 or SEQ ID NO:24;and/or (b) a polypeptide having at least about 80% sequence identity toSEQ ID NO:8, SEQ ID NO:28 or SEQ ID NO:32. In certain embodiments, thepolypeptide is an antibody (e.g., an antibody that specifically binds tothe non-ligand binding membrane proximal region of an extracellulardomain of human Notch1). In certain embodiments, the polypeptidecomprises a polypeptide having at least about 85%, at least about 90%,at least about 95%, at least about 98%, or about 100% sequence identityto SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:8, SEQ ID NO:28 or SEQ IDNO:32. In certain embodiments, the polypeptide comprises a heavy chainvariable region and/or a light chain variable region of the 52M51antibody. In some embodiments, the polypeptide comprises a heavy chainvariable region and/or a light chain variable region of a humanized52M51 antibody. In some embodiment, the polypeptide comprises a heavychain variable region and/or a light chain variable region of antibody52R43.

In another aspect, the invention provides a polynucleotide moleculeencoding any of the antibodies and/or polypeptides of the aforementionedaspects, as well as other aspects/embodiments as described herein. Insome embodiments, an expression vector comprises the polynucleotidemolecule. In other embodiments, a host cell comprises the expressionvector. In some embodiments, a host cell comprises the polynucleotidemolecule. In some embodiments, the host cell is cell line or a hybridomacell line. In certain embodiments, the hybridoma cell line produces the52M51 antibody or a humanized 52M51 antibody.

In a further aspect, the invention provides a method of inhibitingactivity of Notch1 in a cell, comprising contacting the cell with aneffective amount of any of the antibodies or polypeptides described inthe aforementioned aspects and embodiments, as well as otheraspects/embodiments described elsewhere herein. In certain embodiments,the cell is a tumor cell.

In another aspect, the invention provides a method of inhibiting thegrowth of a tumor in a subject, the method comprising administering tothe subject a therapeutically effective amount of any of the antibodiesor polypeptides described in the aforementioned aspects and embodiments,as well as other aspects/embodiments described elsewhere herein. In someembodiments, the tumor comprises cancer stem cells. In some embodiments,the methods comprise targeting the cancer stem cells with theantibodies. In certain embodiments, the methods comprise reducing thefrequency of cancer stem cells in a tumor, reducing the number of cancerstem cells in a tumor, reducing the tumorigenicity of a tumor, and/orreducing the tumorigenicity of a tumor by reducing the number orfrequency of cancer stem cells in the tumor. In some embodiments, themethods comprise inhibiting the activity of a Notch1 receptor and/orinhibiting growth of a tumor. In certain embodiments, the tumor isselected from the group consisting of a breast tumor, colorectal tumor,hepatic tumor, renal tumor, lung tumor, pancreatic tumor, ovarian tumor,prostate tumor and head and neck tumor.

In another aspect, the present invention provides methods of treatingcancer in a subject. In some embodiments, the method comprisesadministering to a subject a therapeutically effective amount of any ofthe antibodies or polypeptides described in the aforementioned aspectsand/or embodiments, as well as other aspects/embodiments describedelsewhere herein. In some embodiments, the cancer to be treated isbreast cancer, colorectal cancer, hepatic cancer, kidney cancer, livercancer, lung cancer, pancreatic cancer, gastrointestinal cancer,melanoma, ovarian cancer, prostate cancer, cervical cancer, bladdercancer, glioblastoma, and head and neck cancer. In certain embodimentsof each of the aforementioned aspects or embodiments, as well as otheraspects and/or embodiments described elsewhere herein, the method oftreating cancer comprises inhibiting tumor growth.

In an additional aspect, the invention provides a method of inhibitinggrowth of a tumor in a subject, the method comprising administering tothe subject a therapeutically effective amount of an antibody thatspecifically binds to a non-ligand binding membrane proximal region ofan extracellular domain of human Notch1, wherein binding inhibitsactivity of Notch1.

In a further aspect, the invention provides a method of reducing thetumorigenicity of a tumor that comprises cancer stem cells by reducingthe frequency or number of cancer stem cells in the tumor, the methodcomprising contacting the tumor with an effective amount of an antibodythat inhibits the activity of Notch1.

In certain embodiments of each of the aforementioned aspects and/orembodiments, as well as other aspects or embodiments described herein,the methods further comprise administering to the subject at least oneadditional anti-cancer and/or therapeutic agent. In certain embodimentsof each of the aforementioned aspects or embodiments, as well as otheraspects and/or embodiments described elsewhere herein, the antibody orpolypeptide is administered to a subject in combination with anadditional treatment for cancer. In certain embodiments, the additionaltreatment for cancer comprises radiation therapy, chemotherapy, and/oran additional antibody therapeutic. In certain embodiments, thechemotherapy comprises taxol, irinotecan, gemcitabine and/oroxaliplatin. In certain embodiments, the additional antibody therapeuticis an antibody that specifically binds a second human Notch receptor(e.g., Notch1) or a human Notch receptor ligand (e.g., DLL4 or JAG1). Incertain embodiments, the additional antibody therapeutic is an antibodythat specifically binds VEGF. In certain embodiments, the subjecttreated is a human.

The invention further provides a method of treating cancer in a human,wherein the cancer comprising cancer stem cells is not characterized byoverexpression by the cancer stem cell of one or more Notch receptors,comprising administering to the human a therapeutically effective amountof an antibody which binds to a membrane proximal region of theextracellular domain of a Notch1 receptor and blocks ligand activationof a Notch1 receptor.

The invention further provides a method of treating cancer in a humancomprising administering to the human therapeutically effective amountsof (a) a first antibody which binds a Notch1 receptor and inhibitsgrowth of cancer stem cells which overexpress Notch receptors; and (b) asecond antibody which binds a Notch receptor and blocks ligandactivation of a Notch receptor.

The invention also provides another method of treating cancer, whereinthe cancer is selected from the group consisting of breast, colon,pancreatic, prostate, lung, rectal and colorectal cancer, comprisingadministering a therapeutically effective amount of an antibody thatblocks ligand activation of a Notch1 receptor.

The invention additionally provides: a humanized antibody which bindsNotch1 and blocks ligand activation of a Notch1 receptor; a compositioncomprising the humanized antibody and a pharmaceutically acceptablecarrier; and an immunoconjugate comprising the humanized antibodyconjugated with a cytotoxic agent.

Moreover, the invention provides an isolated polynucleotide encoding thehumanized antibody; a vector comprising the nucleic acid; a host cellcomprising the nucleic acid or the vector; as well as a process ofproducing the humanized antibody comprising culturing a host cellcomprising the nucleic acid so that the nucleic acid is expressed and,optionally, further comprising recovering the humanized antibody fromthe host cell culture (e.g., from the host cell culture medium).

The invention further pertains to an immunoconjugate comprising anantibody that binds Notch conjugated to one or more calicheamicinmolecules, and the use of such conjugates for treating Notch expressingcancer, e.g., a cancer in which cancer stem cells overexpress Notch.

Examples of solid tumors that can be treated using a therapeuticcomposition of the instant invention, for example, an antibody thatbinds a membrane promixal region of the extracellular domain of a Notch1receptor include, but are not limited to, sarcomas and carcinomas suchas fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but also eachmember of the group individually and all possible subgroups of the maingroup, and also the main group absent one or more of the group members.The present invention also envisions the explicit exclusion of one ormore of any of the group members in the claimed invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Identification of Antibodies Targeting the Membrane ProximalRegion of Notch that Inhibit Notch Signaling.

(A) Schematic of the Notch receptor and 52M antigen region. The 52Mantigen includes the area of the Notch1 receptor subject to cleavage byfurin during maturation of the receptor and cleavage by ADAM (ADisintegrin and Metalloprotease) proteases following ligand binding.Subsequent processing by gamma-secretase causes the release of theintracellular domain (ICD) of Notch that activates gene transcription inthe nucleus. (B) Luciferase levels (y-axis) derived from Notch1-Helacells cultured in the presence of a soluble Notch ligand (hDLL4-fc) andNotch1 receptor antibodies. Results from non-transfected (NT) cells withand without hDLL4-Fc are shown on the far left of the x-axis. 52M Notch1receptor antibodies are shown along the x-axis and compared to DBZ, aNotch gamma-secretase inhibitor (GSI), and 21M18, an anti-DLL4 antibody.Notch1 receptor antibodies 52M51, 52M63, 52M74 and 52M80 allsignificantly inhibited Notch signaling as indicated by a decrease inluciferase activity. (C) Luciferase levels (y-axis) derived fromNotch1-Hela cells cultured in the presence of a soluble Notch ligand(hDLL4-fc) and Notch1 receptor antibodies. Results from non-transfected(NT) cells with and without hDLL4-Fc are shown on the far left of thex-axis. 52M51 murine hybridoma derived antibody and humanized variant52M51-H4/L3 are shown along the x-axis in various concentrations asindicated. Both the parental murine antibody 52M51 and the humanizedvariant significantly inhibited Notch signaling as indicated by adecrease in luciferase activity. (D) Western blot analysis of ICDformation after ligand-mediated stimulation of Notch1 expressing Helacells. Minimal ICD is produced in the absence of DLL4 ligand (-DLL4),but formation is stimulated by the presence of DLL4. Antibodies 52M51,52M63, 52M74, and 52M80 reduce ICD formation to background levelsdespite the presence of DLL4.

FIG. 2: Notch1 Receptor Antibody 52M51 Inhibits Tumor Formation In vivo.

(A) NOD/SCID mice injected with C8 colon tumor cells were treated withcontrol antibody (squares) or anti-Notch1 antibody 52M51 (triangles),and tumor volume (y-axis, mm³) was measured across time (x-axis, days).Treatment with 52M51 antibodies significantly (p=0.0006) inhibited tumorgrowth compared to control. (B) Individual tumor volume measurementsfrom animals in (A) measured on days 48 and 55 for control (left) versus52M51 (right) treated mice. A line demarcates the average of eachexperimental group. (C) NOD/SCID mice injected with PE13 breast tumorcells were treated with control antibody (black squares) or anti-Notch1antibodies that do not inhibit Notch signaling as shown in FIG. 1B: 52M1(black triangles) and 52M2 (grey circles). Tumor volume (y-axis, mm³)was measured across time (x-axis, days). Treatment with 52M1 and 52M2failed to effect tumor growth when compared to control treated animals.(D) NOD/SCID mice injected with PE13 breast tumor cells were treatedwith control antibody (squares) or anti-Notch1 antibody 52M8 (triangles)that does not inhibit Notch signaling as shown in FIG. 1B. Tumor volume(y-axis, mm³) was measured across time (x-axis, days). Treatment with52M8 failed to effect tumor growth when compared to control treatedanimals.

FIG. 3: Anti-Notch1 Receptor Antibody 52R43Inhibits Tumor Growth In vivo

(A) NOD/SCID mice injected with M2 melanoma tumor cells were treatedwith control antibody (squares) or anti-Notch1 antibody 52R43 (circles),and tumor volume (y-axis, mm³) was measured across time (x-axis, days).(B) NOD/SCID mice injected with Lu24 lung tumor cells were treated withcontrol antibody (squares) or anti-Notch1 antibody 52R43 (circles), andtumor volume (y-axis, mm³) was measured across time (x-axis, days). (C)NOD/SCID mice injected with PN8 pancreatic tumor cells were treated withcontrol antibody (squares) or anti-Notch1 antibody 52R43 (circles), andtumor volume (y-axis, mm³) was measured across time (x-axis, days). (D)NOD/SCID mice injected with T1 breast tumor cells were treated withcontrol antibody (squares), anti-Notch1 antibody 52R43 (closed circles),taxol (triangles) or 52R43 and taxol (open circles) and tumor volume(y-axis, mm³) was measured across time (x-axis, days).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel agents, including, but not limitedto polypeptides such as antibodies, that bind one or more human Notchreceptors. The Notch-binding agents include antagonists of the humanNotch receptor(s). Related polypeptides and polynucleotides,compositions comprising the Notch-binding agents, and methods of makingthe Notch-binding agents are also provided. Methods of using the novelNotch-binding agents, such as methods of inhibiting tumor growth and/ortreating cancer, are further provided.

The present invention further identifies molecules (e.g., antibodies)that specifically bind to a non-ligand binding membrane proximal regionof the extracellular domain of a human Notch1 receptor and inhibit tumorgrowth in vivo. The ligand binding region of Notch, which is necessaryand sufficient for ligand binding, has been identified as EGF repeats 11and 12, suggesting this region of the Notch receptor is important inNotch signaling and tumorigenesis (Rebay et al., 1991, Cell 67:687; Leiet al., 2003, Dev. 130:6411; Hambleton et al., 2004, Structure 12:2173).Unexpectedly, antibodies that bind outside the ligand binding domain ofthe extracellular domain of human Notch receptor have been found toinhibit tumor cell growth in vivo (see U.S. Patent Publication No.2008/0131434, incorporated by reference herein in its entirety). Thus,antibodies that bind outside the ligand binding domain of theextracellular domain of one or more of the human Notch receptors—Notch1,Notch2, Notch3, and Notch4—have value as potential cancer therapeutics.

Monoclonal antibodies that specifically bind to the membrane proximalregion of the extracellular domain of a Notch1, including the monoclonalantibody 52M51, have now been identified (Example 1). Humanized 52M51antibodies have also been generated (Example 2). Several of theantibodies, including 52M51 and a humanized variant of 52M51, inhibitligand-induced Notch1 signaling (Example 3 and FIGS. 1B and C), despitebinding to Notch1 in a region outside of the ligand-binding region. Theability of several of the antibodies to inhibit formation of the Notchintracellular domain (ICD) has also now been demonstrated (Example 3 andFIG. 1D). 52M51 has been found to inhibit tumor cell growth in vivo in axenograft model (Example 5 and FIGS. 2A and B). In addition, anotherantibody 52R43 has been found to inhibit tumor cell growth in vivo inmultiple xenograft models (Example 7 and FIG. 3A-D).

DEFINITIONS

An “antagonist” of a Notch receptor as used herein is a term thatincludes any molecule that partially or fully blocks, inhibits, orneutralizes a biological activity of the Notch pathway. Suitableantagonist molecules specifically include antagonist antibodies orantibody fragments. The term “antagonist” is used herein to include anymolecule that partially or fully blocks, inhibits, or neutralizes theexpression of a Notch receptor.

The term “antibody” is used to mean an immunoglobulin molecule thatrecognizes and specifically binds to a target, such as a protein,polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing etc., through at least one antigenrecognition site within the variable region of the immunoglobulinmolecule. As used herein, the term encompasses intact polyclonalantibodies, intact monoclonal antibodies, antibody fragments (such asFab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv) mutants,multispecific antibodies such as bispecific antibodies generated from atleast two intact antibodies, monovalent or monospecific antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be any of the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules such as toxins, radioisotopes, etc.

As used herein, the term “antibody fragment” refers to a portion of anintact antibody and refers to the antigenic determining variable regionsof an intact antibody. Examples of antibody fragments include, but arenot limited to Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies,single chain antibodies, and multispecific antibodies formed fromantibody fragments.

An “Fv antibody” refers to the minimal antibody fragment that contains acomplete antigen-recognition and -binding site either as two-chains, inwhich one heavy and one light chain variable domain form a non-covalentdimer, or as a single-chain (scFv), in which one heavy and one lightchain variable domain are covalently linked by a flexible peptide linkerso that the two chains associate in a similar dimeric structure. In thisconfiguration the complementary determining regions (CDRs) of eachvariable domain interact to define the antigen-binding specificity ofthe Fv dimer Alternatively a single variable domain (or half of an Fv)can be used to recognize and bind antigen, although generally with loweraffinity.

A “monoclonal antibody” as used herein refers to homogenous antibodypopulation involved in the highly specific recognition and binding of asingle antigenic determinant, or epitope. This is in contrast topolyclonal antibodies that typically include different antibodiesdirected against different antigenic determinants. The term “monoclonalantibody” encompasses both intact and full-length monoclonal antibodiesas well as antibody fragments (e.g., Fab, Fab′, F(ab′)2, Fv), singlechain (scFv) mutants, fusion proteins comprising an antibody portion,and any other modified immunoglobulin molecule comprising an antigenrecognition site. Furthermore, “monoclonal antibody” refers to suchantibodies made in any number of manners including but not limited to byhybridoma, phage selection, recombinant expression, and transgenicanimals.

As used herein, the term “humanized antibody” refers to forms ofnon-human (e.g., murine) antibodies that are specific immunoglobulinchains, chimeric immunoglobulins, or fragments thereof that containminimal non-human sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregions (CDRs) are replaced by residues from a CDR of a non-humanspecies (e.g., mouse., rat, rabbit, hamster, etc.) that have the desiredspecificity, affinity, and/or capability. In some instances, the Fvframework region (FR) residues of a human immunoglobulin are replacedwith the corresponding residues in an antibody from a non-human speciesthat has the desired specificity, affinity, and/or capability. Thehumanized antibody can be further modified by the substitution ofadditional residues either in the Fv framework region and/or within thereplaced non-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all, or substantially all, of the CDRregions that correspond to the non-human immunoglobulin whereas all, orsubstantially all, of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. No. 5,225,539, hereinincorporated by reference.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, 5th ed., 1991, National Institutesof Health, Bethesda Md.); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Al-lazikani et al., 1997, J.Molec. Biol. 273:927-948). In addition, combinations of these twoapproaches are sometimes used in the art to determine CDRs.

The term “human antibody” as used herein means an antibody produced by ahuman or an antibody having an amino acid sequence corresponding to anantibody produced by a human made using any of the techniques known inthe art. This definition of a human antibody includes intact orfull-length antibodies, fragments thereof, and/or antibodies comprisingat least one human heavy and/or light chain polypeptide such as, forexample, an antibody comprising murine light chain and human heavy chainpolypeptides.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g., mouse, rat, rabbit, etc.) with the desiredspecificity, affinity, and/or capability, while the constant regions arehomologous to the sequences in antibodies derived from another species(usually human) to avoid eliciting an immune response in that species.The term chimeric antibody includes monovalent, divalent and polyvalentantibodies.

The term “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids (also referredto as linear epitopes) are typically retained upon protein denaturing,whereas epitopes formed by tertiary folding (also referred to asconformational epitopes) are typically lost upon protein denaturing. Anepitope typically includes at least 3, and more usually, at least 5 or8-10 amino acids in a unique spatial conformation.

That an antibody “selectively binds” or “specifically binds” to anepitope or receptor means that the antibody reacts or associates morefrequently, more rapidly, with greater duration, with greater affinity,or with some combination of the above to the epitope or receptor thanwith alternative substances, including unrelated proteins. “Selectivelybinds” or “specifically binds” means, for instance, that an antibodybinds to a protein with a K_(D) of about 0.1 mM or less, at times about1 μM or less, at times about 0.1 μM or less and at times about 0.01 μMor less. Because of the sequence identity between homologous proteins indifferent species, specific binding can include an antibody thatrecognizes a Notch receptor in more than one species. It is understoodthat, in certain embodiments, an antibody or binding moiety thatspecifically binds to a first target may or may not specifically bind toa second target. As such, “specific binding” does not necessarilyrequire (although it can include) exclusive binding, i.e. binding to asingle target. Generally, but not necessarily, reference to bindingmeans specific binding.

Competition between antibodies is determined by an assay in which theimmunoglobulin under study inhibits specific binding of a referenceantibody to a common antigen. Numerous types of competitive bindingassays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242-253 (1983)); solid phase directbiotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619(1986)); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow and Lane, “Antibodies, A Laboratory Manual,”Cold Spring Harbor Press (1988)); solid phase direct label RIA using¹²⁵I label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solidphase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552(1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol.32:77-82 (1990)). Typically, such an assay involves the use of purifiedantigen bound to a solid surface or cells bearing either of these, anunlabeled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur. Usually, when a competing antibody is present in excess, it willinhibit specific binding of a reference antibody to a common antigen byat least 50 or 75%.

The terms “isolated” or “purified” refer to material that issubstantially or essentially free from components that normallyaccompany it in its native state. Purity and homogeneity are typicallydetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography. A protein(e.g., an antibody) or nucleic acid that is the predominant speciespresent in a preparation is substantially purified. In particular, insome embodiments, an isolated nucleic acid comprising a gene isseparated from open reading frames that naturally flank the gene andencode proteins other than the protein encoded by the gene. An isolatedantibody is separated from other non-immunoglobulin proteins and fromother immunoglobulin proteins with different antigen bindingspecificities. It can also mean that the nucleic acid or protein is atleast 85% pure, at least 95% pure, and in some embodiments, at least 99%pure.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth. Examples of cancerinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia. More particular examples of such cancers include, but arenot limited to, squamous cell cancer, small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung, squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial or uterine carcinoma, salivarygland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvalcancer, thyroid cancer, hepatic carcinoma and various types of head andneck cancer.

“Tumor” and “neoplasm” as used herein refer to any mass of tissue thatresult from excessive cell growth or proliferation, either benign(noncancerous) or malignant (cancerous) including pre-cancerous lesions.

The terms “proliferative disorder” and “proliferative disease” refer todisorders associated with abnormal cell proliferation such as cancer.

“Metastasis” as used herein refers to the process by which a cancerspreads or transfers from the site of origin to other regions of thebody with the development of a similar cancerous lesion at the newlocation. A “metastatic” or “metastasizing” cell is one that losesadhesive contacts with neighboring cells and migrates via thebloodstream or lymph from the primary site of disease to invadeneighboring body structures.

The terms “cancer stem cell” or “tumor stem cell” or “solid tumor stemcell” are used interchangeably herein and refer to a population of cellsfrom a solid tumor that: (1) have extensive proliferative capacity; 2)are capable of asymmetric cell division to generate one or more kinds ofdifferentiated progeny with reduced proliferative or developmentalpotential; and (3) are capable of symmetric cell divisions forself-renewal or self-maintenance. These properties of “cancer stemcells” or “tumor stem cells” or “solid tumor stem cells” confer on thosecancer stem cells the ability to form palpable tumors upon serialtransplantation into an immunocompromised mouse compared to the majorityof tumor cells that fail to form tumors. Cancer stem cells undergoself-renewal versus differentiation in a chaotic manner to form tumorswith abnormal cell types that can change over time as mutations occur.

The terms “cancer cell” or “tumor cell” refer to the total population ofcells derived from a tumor including both non-tumorigenic cells, whichcomprise the bulk of the tumor cell population, and tumorigenic stemcells (cancer stem cells).

As used herein “tumorigenic” refers to the functional features of asolid tumor stem cell including the properties of self-renewal (givingrise to additional tumorigenic cancer stem cells) and proliferation togenerate all other tumor cells (giving rise to differentiated and thusnon-tumorigenic tumor cells) that allow solid tumor stem cells to form atumor.

As used herein, the “tumorigenicity” of a tumor refers to the ability ofa random sample of cells from the tumor to form palpable tumors uponserial transplantation into immunocompromised mice.

As used herein, the terms “stem cell cancer marker” or “cancer stem cellmarker” or “tumor stem cell marker” or “solid tumor stem cell marker”refer to a gene or genes or a protein, polypeptide, or peptide expressedby the gene or genes whose expression level, alone or in combinationwith other genes, is correlated with the presence of tumorigenic cancercells compared to non-tumorigenic cells. The correlation can relate toeither an increased or decreased expression of the gene (e.g., increasedor decreased levels of mRNA or the peptide encoded by the gene).

The terms “cancer stem cell gene signature” or “tumor stem cell genesignature” or “cancer stem cell signature” are used interchangeablyherein to refer to gene signatures comprising genes differentiallyexpressed in cancer stem cells compared to other cells or population ofcells, for example normal breast epithelial tissue. In some embodimentsthe cancer stem cell gene signatures comprise genes differentiallyexpressed in cancer stem cells versus normal breast epithelium by a foldchange, for example by 2 fold reduced and/or elevated expression, andfurther limited by using a statistical analysis such as, for example, bythe P value of a t-test across multiple samples. In another embodiment,the genes differentially expressed in cancer stem cells are divided intocancer stem cell gene signatures based on the correlation of theirexpression with a chosen gene in combination with their fold orpercentage expression change. Cancer stem cell signatures are predictiveboth retrospectively and prospectively of an aspect of clinicalvariability, including but not limited to, metastasis and death.

The term “genetic test” as used herein refers to procedures whereby thegenetic make-up of a patient or a patient tumor sample is analyzed. Theanalysis can include detection of DNA, RNA, chromosomes, proteins ormetabolites to detect heritable or somatic disease-related genotypes orkaryotypes for clinical purposes.

As used herein, the terms “biopsy” or “biopsy tissue” refer to a sampleof tissue or fluid that is removed from a subject for the purpose ofdetermining if the sample contains cancerous tissue. In someembodiments, biopsy tissue or fluid is obtained because a subject issuspected of having cancer. The biopsy tissue or fluid is then examinedfor the presence or absence of cancer.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, including humans.

“Pharmaceutically acceptable excipient, carrier or adjuvant” or“acceptable pharmaceutical carrier” refers to an excipient, carrier oradjuvant that can be administered to a subject, together with at leastone antibody of the present disclosure, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the antibody. Inaddition, a “pharmaceutically acceptable carrier” does not trigger animmune response in a recipient subject. Examples include, but are notlimited to, any of the standard pharmaceutical carriers such as aphosphate buffered saline solution, water, and various oil/wateremulsions. Some diluents for aerosol or parenteral administration arephosphate buffered saline or normal (0.9%) saline.

“Pharmaceutically acceptable vehicle” refers to a diluents, adjuvant,excipient, or carrier with which at least one antibody of the presentdisclosure is administered.

The term “effective amount” or “therapeutically effective amount” or“therapeutic effect” refers to an amount of an antibody, polypeptide,polynucleotide, small organic molecule, or other drug effective to“treat” a disease or disorder in a subject or mammal In the case ofcancer, the therapeutically effective amount of the drug has atherapeutic effect and as such can reduce the number of cancer cells;decrease tumorigenicity, tumorigenic frequency or tumorigenic capacity;reduce the number or frequency of cancer stem cells; reduce the tumorsize; inhibit or stop cancer cell infiltration into peripheral organsincluding, for example, the spread of cancer into soft tissue and bone;inhibit and stop tumor metastasis; inhibit and stop tumor growth;relieve to some extent one or more of the symptoms associated with thecancer; reduce morbidity and mortality; improve quality of life; or acombination of such effects. To the extent the agent, for example anantibody, prevents growth and/or kills existing cancer cells, it can bereferred to as cytostatic and/or cytotoxic.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent or slow the development of a targeted pathologiccondition or disorder. Thus those in need of treatment include thosealready with the disorder; those prone to have the disorder; and thosein whom the disorder is to be prevented. A subject is successfully“treated” according to the methods of the present invention if thepatient shows one or more of the following: a reduction in the number ofor complete absence of cancer cells; a reduction in the tumor size;inhibition of or an absence of cancer cell infiltration into peripheralorgans including the spread of cancer into soft tissue and bone;inhibition of or an absence of tumor metastasis; inhibition or anabsence of tumor growth; relief of one or more symptoms associated withthe specific cancer; reduced morbidity and mortality; improvement inquality of life; reduction in tumorigenicity; reduction in the number orfrequency of cancer stem cells; or some combination of effects.

As used herein, the terms “polynucleotide” or “nucleic acid” refer to apolymer composed of a multiplicity of nucleotide units (ribonucleotideor deoxyribonucleotide or related structural variants) linked viaphosphodiester bonds, including but not limited to, DNA or RNA. The termencompasses sequences that include any of the known base analogs of DNAand RNA.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activities or functionalproperties (e.g., enzymatic activity, ligand binding, signaltransduction, immunogenicity, etc.) of the full-length polypeptide orfragment are retained. The term also encompasses the coding region of astructural gene and the sequences located adjacent to the coding regionon both the 5′ and 3′ ends for a distance of about 1 kb or more oneither end such that the gene corresponds to the length of thefull-length mRNA. The term “gene” encompasses both cDNA and genomicforms of a gene.

The term “recombinant” when used with reference to a cell, nucleic acid,protein or vector indicates that the cell, nucleic acid, protein orvector has been modified by the introduction of a heterologous nucleicacid or protein, the alteration of a native nucleic acid or protein, orthat the cell is derived from a cell so modified. Thus, e.g.,recombinant cells express genes that are not found within the native(non-recombinant) form of the cell or express native genes that areoverexpressed or otherwise abnormally expressed such as, for example,expressed as non-naturally occurring fragments or splice variants. Bythe term “recombinant nucleic acid” herein is meant nucleic acid,originally formed in vitro, in general, by the manipulation of nucleicacid, e.g., using polymerases and endonucleases, in a form not normallyfound in nature. In this manner, operably linkage of different sequencesis achieved. Thus an isolated nucleic acid, in a linear form, or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined, are both considered recombinant for the purposes ofthis invention. It is understood that once a recombinant nucleic acid ismade and introduced into a host cell or organism, it will replicatenon-recombinantly, i.e., using the in vivo cellular machinery of thehost cell rather than in vitro manipulations; however, such nucleicacids, once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposes ofthe invention. Similarly, a “recombinant protein” is a protein madeusing recombinant techniques, i.e., through the expression of arecombinant nucleic acid as depicted above.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Vectorsare often derived from plasmids, bacteriophages, or plant or animalviruses.

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (e.g., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (e.g., RNA or protein), while “down-regulation” or “repression”refers to regulation that decrease production. Molecules (e.g.,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

The terms “polypeptide” or “peptide” or “protein” or “protein fragment”are used interchangeably herein to refer to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine. “Amino acid analogs” refersto compounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an alpha carbon that is bound to a hydrogen,a carboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs can have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. “Amino acid mimetics” refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. “Amino acid variants” refers to amino acidsequences. With respect to particular nucleic acid sequences,conservatively modified variants refers to those nucleic acids whichencode identical or essentially identical amino acid sequences, or wherethe nucleic acid does not encode an amino acid sequence, to essentiallyidentical or associated (e.g., naturally contiguous) sequences. Becauseof the degeneracy of the genetic code, a large number of functionallyidentical nucleic acids encode most proteins. For instance, the codonsGCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at everyposition where an alanine is specified by a codon, the codon can bealtered to another of the corresponding codons described withoutaltering the encoded polypeptide. Such nucleic acid variations are“silent variations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes silent variations of the nucleic acid. It isrecognized that in certain contexts each codon in a nucleic acid (exceptAUG, which is ordinarily the only codon for methionine, and TGG, whichis ordinarily the only codon for tryptophan) can be modified to yield afunctionally identical molecule. Accordingly, silent variations of anucleic acid which encodes a polypeptide is implicit in a describedsequence with respect to the expression product, but not with respect toactual probe sequences.

As to amino acid sequences, it will be recognized that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” including where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. (See, for example, Table1). Guidance concerning which amino acid changes are likely to bephenotypically silent can also be found in Bowie et al., 1990, Science247:1306 1310. Such conservatively modified variants are in addition toand do not exclude polymorphic variants, interspecies homologs, andalleles of the invention. Typically conservative substitutionsinclude: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)). As indicated, changes are typically of aminor nature, such as conservative amino acid substitutions that do notsignificantly affect the folding or activity of the protein.

TABLE 1 Conservative Amino Acid Substitutions Original Amino AcidExemplary Conservative Substitutions Alanine Valine, Isoleucine,Leucine, Glycine, Serine Arginine Lysine, Histidine, Glutamine,Asparagine Asparagine Glutamine, Histidine, Lysine, Arginine AsparticAcid Glutamic Acid, Asparagine Cysteine Serine, Alanine, MethionineGlutamine Asparagine Glutamic Acid Aspartic Acid, Glutamine GlycineProline, Alanine Histidine Asparagine, Glutamine, Lysine, ArginineIsoleucine Leucine, Valine, Methionine, Alanine, Phenylalanine,Norleucine Leucine Norleucine, Isoleucine, Valine, Methionine, Alanine,Phenylalanine Lysine Arginine, Glutamine, Asparagine, HistidineMethionine Leucine, Phenylalanine, Isoleucine, Valine, CysteinePhenylalanine Leucine, Valine, Isoleucine, Alanine, Tyrosine ProlineAlanine, Glycine Serine Threonine Threonine Serine Trytophan Tyrosine,Phenylalanine Tyrosine Tryptophan, Phenylalanine, Threonine, SerineValine Isoleucine, Methionine, Leucine, Phenylalanine, Alanine,Norleucine

As used in the present disclosure and claims, the singular forms “a”,“an”, and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that whenever embodiments are described herein with thelanguage “comprising” otherwise analogous embodiments described in termsof “consisting” and/or “consisting essentially of” are also provided.

CERTAIN EMBODIMENTS OF THE PRESENT INVENTION

The present invention provides compositions and methods for studying,diagnosing, characterizing, and treating cancer. In particular, incertain embodiments, the present invention provides agents, includingantagonists, that bind Notch receptors and methods of using the agentsor antagonists to inhibit tumor growth and treat cancer or otherdiseases in human patients. In certain embodiments, the antagonists areantibodies that specifically bind to a non-ligand binding region of theextracellular domain of a human Notch receptor.

In one aspect, the present invention provides an antibody thatspecifically binds to a non-ligand binding membrane proximal region ofthe extracellular domain of a human Notch1 receptor. In someembodiments, the antibody binds a region of human Notch1 comprisingabout amino acid 1427 to about amino acid 1732. In some embodiments, theantibody binds to a region comprising SEQ ID NO:2. In certainembodiments, the antibody that specifically binds to a non-ligandbinding membrane proximal region of the extracellular domain of at leastone additional Notch receptor.

In some embodiments, the antibody is an antagonist of human Notch1. Incertain embodiments, the antibody inhibits ligand-induced signaling of aNotch1 pathway. In some embodiments, the antibody inhibits the activityof Notch1. In other embodiments the antibody inhibits cleavage of aNotch1 receptor. In some embodiments, the antibody inhibits cleavage ofNotch1 at a site within the membrane proximal region of theextracellular domain. In certain embodiments, the antibody inhibitsrelease or formation of the intracellular domain (ICD) of Notch1. Inother embodiments, the antibody reduces the tumorigenicity of a tumorthat comprises cancer stem cells. In certain embodiments, the antibodyinhibits the growth of a tumor comprising cancer stem cells. In certainembodiments, the antibody inhibits the growth of a tumor.

In certain embodiments, the antibody that specifically binds to amembrane proximal region of the extracellular domain of a human Notch1receptor and inhibits tumor growth is a monoclonal antibody. In certainembodiments, the antibody that specifically binds to a membrane proximalregion of the extracellular domain of a human Notch1 receptor is achimeric antibody, is a humanized antibody, is a human antibody, is anantibody fragment, or is a bispecific antibody. In certain embodiments,the present invention provides a hybridoma producing an antibody thatspecifically binds to a non-ligand binding membrane proximal region ofthe extracellular domain of a human Notch1 receptor and inhibits tumorgrowth.

In another aspect, the invention provides a method of inhibiting thegrowth of a tumor in a subject, the method comprising administering tothe subject a therapeutically effective amount of an antibody thatspecifically binds to a non-ligand binding membrane proximal region ofthe extracellular domain of a human Notch1 receptor protein. In someembodiments, the tumor comprises cancer stem cells. In some embodiments,the methods comprise targeting the cancer stem cells with theantibodies. In certain embodiments, the method of inhibiting growth of atumor comprises administering a therapeutically effective amount of amonoclonal antibody. In certain embodiments, the method of inhibitinggrowth of a tumor comprises administering a therapeutically effectiveamount of a chimeric antibody. In certain embodiments, the method ofinhibiting growth of a tumor comprises administering a therapeuticallyeffective amount of a humanized antibody. In certain embodiments, themethod of inhibiting growth of a tumor comprises administering atherapeutically effective amount of a human antibody.

In certain embodiments, the method of inhibiting growth of a tumorcomprises reducing the frequency of cancer stem cells in the tumor,reducing the number of cancer stem cells in the tumor, reducing thetumorigenicity of the tumor, and/or reducing the tumorigenicity of thetumor by reducing the number or frequency of cancer stem cells in thetumor. In some embodiments, the method of inhibiting growth of a tumorcomprises inhibiting the activity of a Notch1 receptor. In certainembodiments, the tumor includes, but is not limited to, a breast tumor,colorectal tumor, hepatic tumor, renal tumor, lung tumor, pancreatictumor, ovarian tumor, prostate tumor and head and neck tumor.

In another aspect, the present invention provides a method of treatingcancer in a subject in need thereof comprising administering to asubject a therapeutically effective amount of an antibody thatspecifically binds to a non-ligand binding membrane proximal region ofthe extracellular domain of a human Notch1 receptor protein and inhibitstumor growth in the subject. In certain embodiments, the method oftreating cancer comprises administering a therapeutically effectiveamount of a monoclonal antibody. In certain embodiments, the method oftreating cancer comprises administering a therapeutically effectiveamount of a chimeric antibody. In certain embodiments, the method oftreating cancer comprises administering a therapeutically effectiveamount of a humanized antibody. In certain embodiments, the method oftreating cancer comprises administering a therapeutically effectiveamount of a human antibody.

In certain embodiments, the method of treating cancer comprisesadministering a therapeutically effective amount of an antibodyconjugated to a cytotoxic moiety that specifically binds to a non-ligandbinding membrane proximal region of the extracellular domain of a humanNotch1 receptor and inhibits tumor growth. In certain embodiments, themethod of treating cancer comprises administering a therapeuticallyeffective amount of an antibody of any of the aspects and/orembodiments, as well as other aspects and/or embodiments describedherein, in combination with radiation therapy. In certain embodiments,the method of treating cancer comprises administering a therapeuticallyeffective amount of an antibody of any of the aspects and/orembodiments, as well as other aspects and/or embodiments describedherein, in combination with chemotherapy. In certain embodiments, themethod of treating cancer comprises administering a therapeuticallyeffective amount of an antibody that specifically binds to a non-ligandbinding membrane proximal region of the extracellular domain of a humanNotch1 receptor and inhibits tumor growth that are from tumorsincluding, but not limited to, a breast tumor, colorectal tumor, lungtumor, pancreatic tumor, prostate tumor, or a head and neck tumor.

In certain embodiments, the method of treating cancer comprisesidentifying patients in need of treatment using a genetic testcomprising an antibody that specifically binds to a non-ligand bindingmembrane proximal region of the extracellular domain of a human Notch1receptor; and administering a therapeutically effective amount of theantibody to the patients. In certain embodiments, the method of treatingcancer comprises identifying patients in need of treatment with anantibody that specifically binds to a non-ligand binding membraneproximal region of the extracellular domain of a human Notch1 receptorusing a genetic test that detects a cancer stem cell signature, andadministering a therapeutically effective amount of the antibody thatspecifically binds to a non-ligand binding membrane proximal region ofthe extracellular domain of a human Notch1 receptor and inhibits tumorgrowth.

In another aspect, the present invention provides a method ofidentifying a molecule that binds to a non-ligand binding membraneproximal region of the extracellular domain of a human Notch1 receptorand inhibits tumor growth, the method comprising: i) incubating themolecule with a non-ligand binding membrane proximal region of theextracellular domain of a human Notch1 receptor; ii) determining if themolecule binds to the non-ligand binding membrane proximal region of theextracellular domain of the human Notch1 receptor; and iii) determiningif the molecule inhibits tumor growth. In certain embodiments, theinvention provides a method of identifying a molecule that binds to anon-ligand binding membrane proximal region of an extracellular domainof a human Notch1 receptor and inhibits tumor growth, the methodcomprising: i) incubating the molecule with the non-ligand bindingmembrane proximal region of the extracellular domain of a human Notch1receptor comprising SEQ ID NO:2; ii) determining if the molecule bindsto the non-ligand binding membrane proximal region of the extracellulardomain of the human Notch1 receptor comprising SEQ ID NO:2; and iii)determining if the molecule inhibits tumor growth.

In certain embodiments, the present invention provides a pharmaceuticalcomposition comprising an antibody that specifically binds to anon-ligand binding membrane proximal region of the extracellular domainof a human Notch1 receptor and inhibits tumor growth.

In certain embodiments, the present invention provides a method ofmaking an antibody that specifically binds to a non-ligand bindingmembrane proximal region of the extracellular domain of a human Notch1receptor and inhibits tumor growth.

In certain embodiments, the present invention provides an isolatednucleic acid that encodes an antibody that specifically binds to anon-ligand membrane proximal binding region of the extracellular domainof a human Notch1 receptor and inhibits tumor growth.

In certain embodiments, antagonists against a Notch receptor, such asNotch1, act extracellularly to act upon or inhibit the function of theNotch receptor. In certain embodiments, an antagonist of a Notchreceptor is proteinaceous. In some embodiments, proteinaceousantagonists of a Notch1 receptor are antibodies that specifically bindto an extracellular epitope of a Notch1 receptor. Extracellular bindingof an antagonist against a Notch1 receptor can inhibit the signaling ofa Notch receptor by inhibiting intrinsic activation (e.g. kinaseactivity) of a Notch1 receptor and/or by sterically inhibiting theinteraction, for example, of a Notch receptor with one of its ligands.Furthermore, extracellular binding of an antagonist to a Notch receptorcan downregulate cell-surface expression of a Notch receptor such as,for example, by internalization of a Notch receptor and/or decreasingcell surface trafficking of a Notch receptor. Extracellular binding ofan antagonist to a Notch receptor can inhibit cleavage of the Notchreceptor and reduce release of the ICD of Notch.

In some embodiments, antagonists against a Notch receptor bind to aNotch receptor and have one or more of the following effects: inhibitproliferation of tumor cells, trigger cell death directly in tumorcells, or prevent metastasis of tumor cells. In certain embodiments,antagonists of a Notch receptor trigger cell death via a conjugatedtoxin, chemotherapeutic agent, radioisotope, or other such agent. Forexample, an antibody against a Notch receptor is conjugated to a toxinthat is activated in tumor cells expressing the Notch receptor byprotein internalization. In other embodiments, antagonists of a Notchreceptor mediate cell death of a cell expressing the Notch receptor viaantibody-dependent cellular cytotoxicity (ADCC). ADCC involves celllysis by effector cells that recognize the Fc portion of an antibody.Many lymphocytes, monocytes, tissue macrophages, granulocytes andeosinophils, for example, have Fc receptors and can mediate cytolysis(Dillman, 1994, J. Clin. Oncol. 12:1497). In some embodiments, anantagonist of a Notch receptor is an antibody that triggers cell deathof cell expressing a Notch receptor by activating complement-dependentcytotoxicity (CDC). CDC involves binding of serum complement to the Fcportion of an antibody and subsequent activation of the complementprotein cascade, resulting in cell membrane damage and eventual celldeath. Biological activity of antibodies is known to be determined, to alarge extent, by the constant domains or Fc region of the antibodymolecule (Uananue and Benacerraf, Textbook of Immunology, 2nd Edition,Williams & Wilkins, p. 218 (1984)). Antibodies of different classes andsubclasses differ in this respect, as do antibodies of the same subclassbut from different species. Of human antibodies, IgM is the mostefficient class of antibodies to bind complement, followed by IgG1,IgG3, and IgG2 whereas IgG4 appears quite deficient in activating thecomplement cascade (Dillman, 1994, J. Clin. Oncol. 12:1497; Jefferis etal., 1998, Immunol. Rev. 163:59-76). According to the present invention,antibodies of those classes having the desired biological activity areprepared.

The ability of any particular antibody against a Notch receptor tomediate lysis of the target cell by complement activation and/or ADCCcan be assayed. The cells of interest are grown and labeled in vitro;the antibody is added to the cell culture in combination with eitherserum complement or immune cells which can be activated by the antigenantibody complexes. Cytolysis of the target cells is detected, forexample, by the release of label from the lysed cells. In fact,antibodies can be screened using the patient's own serum as a source ofcomplement and/or immune cells. The antibody that is capable ofactivating complement or mediating ADCC in the in vitro test can then beused therapeutically in that particular patient.

In certain embodiments, the Notch-binding agent or antagonist is anantibody that does not have one or more effector functions. Forinstance, in some embodiments, the antibody has no antibody-dependentcellular cytotoxicity (ADCC) activity, and/or no complement-dependentcytoxicity (CDC) activity. In certain embodiments, the antibody does notbind to the Fc receptor and/or complement factors. In certainembodiments, the antibody has no effector function.

In other embodiments, antagonists of a Notch receptor can trigger celldeath indirectly by inhibiting angiogenesis. Angiogenesis is the processby which new blood vessels form from pre-existing vessels and is afundamental process required for normal growth, for example, duringembryonic development, wound healing and in response to ovulation. Solidtumor growth larger than 1-2 mm² also requires angiogenesis to supplynutrients and oxygen without which tumor cells die. Thus in certainembodiments, an antagonist of a Notch receptor targets vascular cellsthat express the Notch receptor including, for example, endothelialcells, smooth muscle cells or components of the extracellular matrixrequired for vascular assembly. In other embodiments, an antagonist of aNotch receptor inhibits growth factor signaling required by vascularcell recruitment, assembly, maintenance or survival.

The present invention provides a variety of polypeptides, including butnot limited to antibodies and fragments of antibodies. In certainembodiments, the polypeptide is isolated. In certain alternativeembodiments, the polypeptide is substantially pure.

In certain embodiments, the polypeptides of the present invention can berecombinant polypeptides, natural polypeptides, or syntheticpolypeptides comprising the sequence of SEQ ID NO:8, SEQ ID NO:14, SEQID NO:24, SEQ ID NO:28, or SEQ ID NO:32 (with or without the indicatedsignal sequences).

The invention provides a polypeptide comprising the heavy chain and/orthe light chain provided in SEQ ID NO:10 and/or SEQ ID NO:4,respectively (with or without the indicated putative signal sequences).In certain embodiments, the polypeptide is an antibody. In certainembodiments, the polypeptide specifically binds a non-ligand bindingmembrane proximal region of the extracellular domain of a human Notch1receptor.

The invention further provides a polypeptide comprising SEQ ID NO:8, SEQID NO:28 or SEQ ID NO:32, and/or SEQ ID NO:14 or SEQ ID NO:24. Incertain embodiments, the polypeptide comprises a variable light chainsequence comprising SEQ ID NO:8 and a variable heavy chain sequencecomprising SEQ ID NO:14. In certain embodiments, the polypeptidecomprises a variable light chain sequence comprising SEQ ID NO:28 and avariable heavy chain sequence comprising SEQ ID NO:24. In certainembodiments, the polypeptide comprises a variable light chain sequencecomprising SEQ ID NO:32 and a variable heavy chain sequence comprisingSEQ ID NO:24. In certain embodiments, the polypeptide is an antibody. Incertain embodiments, the polypeptide specifically binds a non-ligandbinding membrane proximal region of the extracellular domain of a humanNotch1 receptor.

It will be recognized in the art that some amino acid sequences of theinvention can be varied without significant effect of the structure orfunction of the protein. If such differences in sequence arecontemplated, it should be remembered that there will be critical areason the protein which determine activity. Thus, the invention furtherincludes variations of the polypeptides which show substantial activity.Such mutants include deletions, insertions, inversions, repeats, andtype substitutions. Guidance concerning which amino acid changes arelikely to be phenotypically silent can be found in Bowie, J. U., et al.,“Deciphering the Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 1990, 247:1306-1310.

Thus, the fragments, derivatives, or analogs of the polypeptides of theinvention can be: (i) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue (often a conserved amino acid residue) and such substitutedamino acid residue can or cannot be one encoded by the genetic code; or(ii) one in which one or more of the amino acid residues includes asubstituent group; or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol); or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives, and analogs are deemed to be within the scope ofthe teachings herein.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge. Theprevention of aggregation is highly desirable. Aggregation of proteinsnot only results in a loss of activity but can also be problematic whenpreparing pharmaceutical formulations, because they can be immunogenic.(Pinckard et al., Clin. Exp. Immunol. 1967, 2:331-340; Robbins et al.,Diabetes 1987, 36:838-845; Cleland et al. Crit. Rev. Therapeutic DrugCarrier Systems 1993, 10:307-377).

Of course, the number of amino acid substitutions made depends on manyfactors, including those described herein. In certain embodiments, thenumber of substitutions for any given polypeptide will not be more than50, 40, 30, 25, 20, 15, 10 or 3.

The polypeptides of the present invention include the polypeptides ofSEQ ID NO:14 as well as polypeptides which have at least 90% similarity(at certain times at least 90% sequence identity) to the polypeptides ofSEQ ID NO:14 and at least 95% similarity (at certain times at least 95%sequence identity) to the polypeptides of SEQ ID NOs:14, and in stillother embodiments, polypeptide which have at least 96%, 97%, 98%, or 99%similarity (at certain times 96%, 97%, 98%, or 99% sequence identity) tothe polypeptides of SEQ ID NOs:14. The polypeptides of the presentinvention include the polypeptides of SEQ ID NO:8 as well aspolypeptides which have at least 90% similarity (at certain times atleast 90% sequence identity) to the polypeptides of SEQ ID NO:8 and atleast 95% similarity (at certain times at least 95% sequence identity)to the polypeptides of SEQ ID NOs:8, and in still other embodiments,polypeptide which have at least 96%, 97%, 98%, or 99% similarity (atcertain times 96%, 97%, 98%, or 99% sequence identity) to thepolypeptides of SEQ ID NOs:8. As known in the art “similarity” betweentwo polypeptides is determined by comparing the amino acid sequence andits conserved amino acid substitutes of one polypeptide to the sequenceof a second polypeptide.

Fragments or portions of the polypeptides of the present invention canbe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments can be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention can be used tosynthesize full-length polynucleotides of the present invention.

In certain embodiments, a fragment of the proteins of this invention isa portion or all of a protein which is capable of binding to a Notch1receptor protein. This fragment has a high affinity for a Notch receptoror a ligand of a Notch1 receptor. Certain fragments of fusion proteinsare protein fragments comprising at least part of the Notch bindingdomain of the polypeptide agent or antagonist fused to at least part ofa constant region of an immunoglobulin. The affinity is typically in therange of about 10⁻¹¹ to 10⁻¹² M, although the affinity can varyconsiderably with fragments of different sizes, ranging from 10⁻⁷ to10⁻¹³ M. In some embodiments, the fragment is about 10-110 amino acidsin length and comprises the Notch binding domain of the polypeptideagent or antagonist linked to at least part of a constant region of animmunoglobulin.

The polypeptides and analogs can be further modified to containadditional chemical moieties not normally part of the protein. Thederivatized moieties can improve the solubility, the biological halflife and/or absorption of the protein. The moieties can also reduce oreliminate any undesirable side effects of the protein and the like. Anoverview for chemical moieties can be found in Remington'sPharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, Pa.(2000).

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthesis methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host.

In some embodiments of a recombinant method, a DNA sequence isconstructed by isolating or synthesizing a DNA sequence encoding awild-type protein of interest. Optionally, the sequence can bemutagenized by site-specific mutagenesis to provide functional analogsthereof. See, e.g. Zoeller et al., Proc.-Nat. Acad. Sci. USA 1984,81:5662-5066 and U.S. Pat. No. 4,588,585. Another method of constructinga DNA sequence encoding a polypeptide of interest would be by chemicalsynthesis using an oligonucleotide synthesizer. Such oligonucleotidescan be designed based on the amino acid sequence of the desiredpolypeptide and selecting those codons that are favored in the host cellin which the recombinant polypeptide of interest will be produced.

Standard methods can be applied to synthesize an isolated polynucleotidesequence encoding an isolated polypeptide of interest. For example, acomplete amino acid sequence can be used to construct a back-translatedgene. Further, a DNA oligomer containing a nucleotide sequence codingfor the particular isolated polypeptide can be synthesized. For example,several small oligonucleotides coding for portions of the desiredpolypeptide can be synthesized and then ligated. The individualoligonucleotides typically contain 5′ or 3′ overhangs for complementaryassembly.

Once assembled (by synthesis, site-directed mutagenesis, or anothermethod), the mutant DNA sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene is operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

The present invention provides isolated antibodies against a non-ligandbinding membrane proximal region of the extracellular domain of a Notch1receptor. The antibody, or antibody fragment, can be any monoclonal orpolyclonal antibody that specifically recognizes a membrane proximalregion of the extracellular domain of Notch1. In some embodiments, thepresent invention provides monoclonal antibodies, or fragments thereof,that specifically bind to a membrane proximal region of theextracellular domain of a human Notch1 as described herein. In someembodiments, the monoclonal antibodies, or fragments thereof, arechimeric or humanized antibodies that specifically bind to a membraneproximal region of the extracellular domain of a human Notch1 receptoras described herein. In other embodiments, the monoclonal antibodies, orfragments thereof, are human antibodies that specifically bind to amembrane proximal region of the extracellular domain of a human Notch1receptor as described herein.

The antibodies against a membrane proximal region of the extracellulardomain of a Notch1 receptor find use in the experimental, diagnostic andtherapeutic methods described herein. In certain embodiments, theantibodies of the present invention are used to detect the expression ofa Notch1 receptor in biological samples such as, for example, a patienttissue biopsy, pleural effusion, or blood sample. Tissue biopsies can besectioned and protein detected using, for example, immunofluorescence orimmunohistochemistry. Alternatively, individual cells from a sample areisolated, and protein expression detected on fixed or live cells by FACSanalysis. Furthermore, the antibodies can be used on protein arrays todetect expression of a Notch1 receptor, for example, on tumor cells, incell lysates, or in other protein samples. In other embodiments, theantibodies of the present invention are used to inhibit the growth oftumor cells by contacting the antibodies with tumor cells either in invitro cell based assays or in vivo animal models. In still otherembodiments, the antibodies are used to treat cancer in a human patientby administering a therapeutically effective amount of an antibodyagainst a membrane proximal region of the extracellular domain of aNotch1 receptor.

Polyclonal antibodies can be prepared by any known method. Polyclonalantibodies are raised by immunizing an animal (e.g. a rabbit, rat,mouse, goat, donkey, etc.) by multiple subcutaneous or intraperitonealinjections of the relevant antigen (a purified peptide fragment,full-length recombinant protein, fusion protein, etc.) optionallyconjugated to keyhole limpet hemocyanin (KLH), serum albumin, etc.diluted in sterile saline and combined with an adjuvant (e.g., Completeor Incomplete Freund's Adjuvant) to form a stable emulsion. Thepolyclonal antibody is then recovered from blood, ascites and the like,of an animal so immunized Collected blood is clotted, and the serumdecanted, clarified by centrifugation, and assayed for antibody titer.The polyclonal antibodies can be purified from serum or ascitesaccording to standard methods in the art including affinitychromatography, ion-exchange chromatography, gel electrophoresis,dialysis, etc.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein, 1975, Nature 256:495-497. Usingthe hybridoma method, a mouse, hamster, or other appropriate hostanimal, is immunized as described above to elicit the production ofantibodies by lymphocytes that will specifically bind to an immunizingantigen. Alternatively, lymphocytes can be immunized in vitro. Followingimmunization, the lymphocytes are isolated and fused with a suitablemyeloma cell line using, for example, polyethylene glycol, to formhybridoma cells that can then be selected away from unfused lymphocytesand myeloma cells. Hybridomas that produce monoclonal antibodiesdirected specifically against a chosen antigen as determined byimmunoprecipitation, immunoblotting, or by an in vitro binding assaysuch as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay(ELISA) can then be propagated either in vitro culture using standardmethods (Goding, Monoclonal Antibodies: Principles and Practice,Academic Press, 1986) or in vivo as ascites tumors in an animal. Themonoclonal antibodies can then be purified from the culture medium orascites fluid as described for polyclonal antibodies above.

In some embodiments of the present invention, the antibody is anantibody that specifically binds to a non-ligand binding membraneproximal region of the extracellular domain of a human Notch1 receptor.In some embodiments, the antibody comprises a heavy chain variableregion having at least 90% sequence identity to SEQ ID NO:14; and/or alight chain variable region having at least 90% sequence identity to SEQID NO:8. In some embodiments, the antibody comprises a heavy chainvariable region having at least 95% sequence identity to SEQ ID NO:14,and/or a light chain variable region having at least 95% sequenceidentity to SEQ ID NO:8. In some embodiments, the antibody is amonoclonal antibody or antibody fragment.

In certain embodiments, the invention provides an antibody that binds anon-ligand binding membrane proximal region of the extracellular domainof a human Notch1 and comprises a heavy chain CDR1 comprising RGYWIE(SEQ ID NO:15), a heavy chain CDR2 comprising QILPGTGRTNYNEKFKG (SEQ IDNO:16), and/or a heavy chain CDR3 comprising FDGNYGYYAMDY (SEQ IDNO:17). In some embodiments, the antibody further comprises a lightchain CDR1 comprising RSSTGAVTTSNYAN (SEQ ID NO:18), a light chain CDR2comprising GTNNRAP (SEQ ID NO:19), and/or a light chain CDR3 comprisingALWYSNHWVFGGGTKL (SEQ ID NO:20). In some embodiments, the antibodycomprises a heavy chain CDR1 comprising RGYWIE (SEQ ID NO:15), a heavychain CDR2 comprising QILPGTGRTNYNEKFKG (SEQ ID NO:16), and/or a heavychain CDR3 comprising FDGNYGYYAMDY (SEQ ID NO:17); and a light chainCDR1 comprising RSSTGAVTTSNYAN (SEQ ID NO:18), a light chain CDR2comprising GTNNRAP (SEQ ID NO:19), and/or a light chain CDR3 comprisingALWYSNHWVFGGGTKL (SEQ ID NO:20). In some embodiments, the antibodycomprises a heavy chain variable region comprising: (a) a heavy chainCDR1 comprising RGYWIE (SEQ ID NO:15), or a variant thereof comprising1, 2, 3, or 4 amino acid substitutions; (b) a heavy chain CDR2comprising QILPGTGRTNYNEKFKG (SEQ ID NO:16), or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions; and/or (c) a heavychain CDR3 comprising FDGNYGYYAMDY (SEQ ID NO:17), or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions. In other embodiments,the antibody comprises a light chain variable region comprising: (a) alight chain CDR1 comprising RSSTGAVTTSNYAN (SEQ ID NO:18), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) a lightchain CDR2 comprising GTNNRAP (SEQ ID NO:19), or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions; and/or (c) a lightchain CDR3 comprising ALWYSNHWVFGGGTKL (SEQ ID NO:20), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions. In someembodiments, the amino acid substitutions are conservative amino acidsubstitutions.

In some embodiments, the invention provides an antibody, 52M51, producedby the hybridoma cell line deposited with the ATCC under the conditionsof the Budapest Treaty on Aug. 7, 2008 and assigned number PTA-9405. Insome embodiments, the antibody is a humanized version of 52M51. In someembodiments, the antibody is a humanized version of 52M51, “52M51H4L3”,as encoded by the DNA deposited with the ATCC under the conditions ofthe Budapest Treaty on Oct. 15, 2008 and assigned number PTA-9549. Insome embodiments, the antibody is a humanized version of 52M51,“52M51H4L4”. In some embodiments, the invention provides an antibodythat binds to the same epitope as the epitope to which antibody 52M51binds. In other embodiments, the invention provides an antibody thatcompetes with any of the antibodies as described in the aforementionedembodiments and/or aspects, as well as other aspects/embodimentsdescribed elsewhere herein, for specific binding to a non-ligand bindingmembrane proximal region of the extracellular domain of a human Notch1receptor. Pharmaceutical compositions comprising the antibodies andmethods of treating cancer comprising administering therapeuticallyeffective amounts of the antibodies are also provided.

In some embodiments, the invention provides an antibody, 52R43, asencoded by the DNA deposited with the ATCC under the conditions of theBudapest Treaty on Oct. 15, 2008 and assigned number PTA-9548. In someembodiments, the invention provides an antibody that binds to the sameepitope as the epitope to which antibody 52R43 binds. In someembodiments, the invention provides an antibody that comprises one, two,three, four, five and/or six of the CDRs of 52R43. In other embodiments,the invention provides an antibody that competes with 52R43.Pharmaceutical compositions comprising the antibodies and methods oftreating cancer comprising administering therapeutically effectiveamounts of the antibodies are also provided.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. Polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cells, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. Isolated polynucleotides encoding the heavy and light chainsare then cloned into suitable expression vectors are then transfectedinto host cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein. Host cells are screened for monoclonal antibodyproduction and antibodies with the desired specificity are selected.Also, recombinant monoclonal antibodies or fragments thereof of thedesired species can be isolated from phage display libraries asdescribed (McCafferty et al., 1990, Nature, 348:552-554; Clackson etal., 1991, Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol.,222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In some embodiments, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In otherembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Furthermore,site-directed or high-density mutagenesis of the variable region can beused to optimize specificity, affinity, etc. of a monoclonal antibody.

More generally, modified antibodies useful in the present invention maybe obtained or derived from any antibody. Further, the parent orprecursor antibody, or fragment thereof, used to generate the disclosedmodified antibodies may be murine, human, chimeric, humanized, non-humanprimate or primatized. In other embodiments the modified antibodies ofthe present invention can comprise single chain antibody constructs(such as that disclosed in U.S. Pat. No. 5,892,019, which isincorporated herein by reference) having altered constant domains asdescribed herein. Consequently, any of these types of antibodiesmodified in accordance with the teachings herein are compatible withthis invention.

According to the present invention, techniques can be adapted for theproduction of single-chain antibodies specific to a polypeptide of theinvention (see U.S. Pat. No. 4,946,778). In addition, methods can beadapted for the construction of Fab expression libraries (Huse, et al.,1989, Science, 246:1275-1281) to allow rapid and effectiveidentification of monoclonal Fab fragments with the desired specificityfor Notch or derivatives, fragments, analogs or homologs thereof.Antibody fragments that contain the idiotypes to a polypeptide of theinvention may be produced by techniques in the art including, but notlimited to: (a) an F(ab′)₂ fragment produced by pepsin digestion of anantibody molecule; (b) an Fab fragment generated by reducing thedisulfide bridges of an F(ab′)₂ fragment, (c) an Fab fragment generatedby the treatment of the antibody molecule with papain and a reducingagent, and (d) Fv fragments.

Bispecific antibodies are also within the scope of the invention.Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens.

Methods for making bispecific antibodies are known in the art. Forexample, in the present case, one of the binding specificities is for anantigenic polypeptide of the invention (Notch1 or a fragment thereof),while the second binding target is any other antigen, and advantageouslyis a cell surface protein, or receptor or receptor subunit. Recombinantproduction of bispecific antibodies is based on the co-expression of twoimmunoglobulin heavy chain/light chain pairs, where the two heavy chainshave different specificities (Milstein and Cuello, Nature 1983,305:537-539). Because of the random assortment of immunoglobulin heavyand light chains, these hybridomas (quadromas) produce a potentialmixture of ten different antibody molecules, of which only one has thecorrect bispecific structure. The purification of the correct moleculeis usually accomplished by affinity chromatography.

Antibody variable domains with the desired binding specificities can befused to immunoglobulin constant domain sequences. The fusion is with animmunoglobulin heavy chain constant domain, comprising at least part ofthe hinge, CH2 and CH3 regions. The first heavy chain constant region(CH1) containing the site necessary for light chain binding can bepresent in at least one of the fusions. DNA encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are co-transfected into asuitable host organism. Further details of generating bispecificantibodies can be found in Suresh et al., Methods in Enzymology 1986,121:210.

Bispecific antibodies can be prepared as full-length antibodies orantibody fragments. Techniques for generating bispecific antibodies fromantibody fragments have been described in the literature. For example,bispecific antibodies can be prepared using chemical linkage. Inaddition, Brennan et al., Science 1985, 229:81 describe a procedurewherein intact antibodies are proteolytically cleaved to generateF(ab′)₂ fragments.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies (Shalaby et al., J.Exp. Med. 1992, 175:217-225). These methods can be used in theproduction of a fully humanized bispecific antibody F(ab′)₂ molecule.

Antibodies with more than two valencies are also contemplated. Forexample, trispecific antibodies can be prepared (Tutt et al., 1991, J.Immunol. 147:60).

This invention also encompasses bispecific antibodies that specificallyrecognize the membrane proximal region of a extracellular domain of aNotch1 receptor. Bispecific antibodies are antibodies that are capableof specifically recognizing and binding at least two different epitopes.The different epitopes can either be within the same molecule (e.g. thesame Notch1) or on different molecules such that both, for example, theantibodies can specifically recognize and bind a Notch1 receptor, aswell as, for example, 1) an effector molecule on a leukocyte such as aT-cell receptor (e.g. CD3) or Fc receptor (e.g. CD64, CD32, or CD 16) or2) a cytotoxic agent as described in detail below. Bispecific antibodiescan be intact antibodies or antibody fragments. Techniques for makingbispecific antibodies are common in the art (Millstein et al., 1983,Nature, 305:537-539; Brennan et al., 1985, Science, 229:81; Suresh etal, 1986, Methods in Enzymol., 121:120; Traunecker et al., 1991, EMBOJ., 10:3655-3659; Shalaby et al., 1992, J. Exp. Med., 175:217-225;Kostelny et al., 1992, J. Immunol., 148:1547-1553; Gruber et al., 1994,J. Immunol., 152:5368; and U.S. Pat. No. 5,731,168).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in a polypeptide of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG so as to focus cellular defense mechanismsto the cell expressing the particular antigen. Bispecific antibodies canalso be used to direct cytotoxic agents to cells which express aparticular antigen. These antibodies possess an antigen-binding arm andan arm which binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA.

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It iscontemplated that the antibodies can be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins can be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For the purposes of the present invention, it should be appreciated thatmodified antibodies can comprise any type of variable region thatprovides for the association of the antibody with a membrane proximalregion of the extracellular domain of a Notch1 receptor. In this regard,the variable region may comprise or be derived from any type of mammalthat can be induced to mount a humoral response and generateimmunoglobulins against the desired tumor associated antigen. As such,the variable region of the modified antibodies can be, for example, ofhuman, murine, non-human primate (e.g. cynomolgus monkeys, macaques,etc.) or lupine origin. In some embodiments both the variable andconstant regions of the modified immunoglobulins are human. In otherembodiments the variable regions of compatible antibodies (usuallyderived from a non-human source) can be engineered or specificallytailored to improve the binding properties or reduce the immunogenicityof the molecule. In this respect, variable regions useful in the presentinvention can be humanized or otherwise altered through the inclusion ofimported amino acid sequences.

In some embodiments, of the present invention the monoclonal antibodyagainst a membrane proximal region of the extracellular domain of aNotch1 receptor is a humanized antibody. Humanized antibodies areantibodies that contain minimal sequences from non-human (e.g., murine)antibodies within the variable regions. Such antibodies are usedtherapeutically to reduce antigenicity and HAMA (human anti-mouseantibody) responses when administered to a human subject. In practice,humanized antibodies are typically human antibodies with minimum to nonon-human sequences. A human antibody is an antibody produced by a humanor an antibody having an amino acid sequence corresponding to anantibody produced by a human.

Humanized antibodies can be produced using various techniques known inthe art. An antibody can be humanized by substituting the CDR of a humanantibody with that of a non-human antibody (e.g. mouse, rat, rabbit,hamster, etc.) having the desired specificity, affinity, and/orcapability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al.,1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science,239:1534-1536). The humanized antibody can be further modified by thesubstitution of additional residue either in the Fv framework regionand/or within the replaced non-human residues to refine and optimizeantibody specificity, affinity, and/or capability.

In some embodiments of the present invention, the antibody is ahumanized antibody which specifically binds to a non-ligand bindingmembrane proximal region of the extracellular domain of a human Notch1receptor. In some embodiments, the antibody comprises a heavy chainvariable region having at least 90% sequence identity to SEQ ID NO:24;and/or a light chain variable region having at least 90% sequenceidentity to SEQ ID NO:28 or SEQ ID NO:32. In some embodiments, theantibody comprises a heavy chain variable region having at least 95%sequence identity to SEQ ID NO:24, and/or a light chain variable regionhaving at least 95% sequence identity to SEQ ID NO:28 or SEQ ID NO:32.

In some embodiments, the humanized antibody comprises a heavy chainvariable region of SEQ ID NO:24, and a light chain variable region ofSEQ ID NO:28. In some embodiments, the humanized antibody comprises aheavy chain variable region of SEQ ID NO:24, and a light chain variableregion of SEQ ID NO:32.

Human antibodies can be directly prepared using various techniques knownin the art. Immortalized human B lymphocytes immunized in vitro orisolated from an immunized individual that produces an antibody directedagainst a target antigen can be generated (See, for example, Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985); Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat.No. 5,750,373). Also, the human antibody can be selected from a phagelibrary, where that phage library expresses human antibodies (Vaughan etal., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS,95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Markset al., 1991, J. Mol. Biol., 222:581). Humanized antibodies can also bemade in transgenic mice containing human immunoglobulin loci that arecapable, upon immunization, of producing the full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray into such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. (See, for example, Jakobovitset al., 1993, Proc. Natl. Acad. Sci. USA, 90:2551; Jakobovits et al.,1993, Nature, 362:255-258; Bruggemann et al., 1993, Year in Immuno.7:33; U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and 5,661,016

Alternatively, phage display technology can be used to produce humanantibodies and antibody fragments in vitro, from immunoglobulin variable(V) domain gene repertoires from unimmunized donors. According to thistechnique, antibody V domain genes are cloned in-frame into either amajor or minor coat protein gene of a filamentous bacteriophage, such asM13 or fd, and displayed as functional antibody fragments on the surfaceof the phage particle. Because the filamentous particle contains asingle-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. Thus, the phagemimics some of the properties of the B-cell. Phage display can beperformed in a variety of formats. Several sources of V-gene segmentscan be used for phage display. A diverse array of anti-oxazoloneantibodies have been isolated from a small random combinatorial libraryof V genes derived from the spleens of immunized mice. A repertoire of Vgenes from unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can be isolated.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

It will be appreciated that grafting the entire non-human variabledomains onto human constant regions will produce “classic” chimericantibodies. In the context of the present application the term “chimericantibodies” will be held to mean any antibody wherein the immunoreactiveregion or site is obtained or derived from a first species and theconstant region (which may be intact, partial or modified in accordancewith this invention) is obtained from a second species. In someembodiments, the antigen binding region or site will be from a non-humansource (e.g. mouse) and the constant region is human. While theimmunogenic specificity of the variable region is not generally affectedby its source, a human constant region is less likely to elicit animmune response from a human subject than would the constant region froma non-human source.

The variable domains in both the heavy and light chains are altered byat least partial replacement of one or more CDRs and, if necessary, bypartial framework region replacement and sequence changing. Although theCDRs may be derived from an antibody of the same class or even subclassas the antibody from which the framework regions are derived, it isenvisaged that the CDRs will be derived from an antibody of differentclass and preferably from an antibody from a different species. It mustbe emphasized that it may not be necessary to replace all of the CDRswith the complete CDRs from the donor variable region to transfer theantigen binding capacity of one variable domain to another. Rather, itmay only be necessary to transfer those residues that are necessary tomaintain the activity of the antigen binding site. Given theexplanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and5,693,762, it will be well within the art, either by carrying outroutine experimentation or by trial and error testing to obtain afunctional antibody with reduced immunogenicity.

Alterations to the variable region notwithstanding, it will beappreciated that the modified antibodies of this invention will compriseantibodies, or immunoreactive fragments thereof, in which at least afraction of one or more of the constant region domains has been deletedor otherwise altered so as to provide desired biochemicalcharacteristics such as increased tumor localization or reduced serumhalf-life when compared with an antibody of approximately the sameimmunogenicity comprising a native or unaltered constant region. In someembodiments, the constant region of the modified antibodies willcomprise a human constant region. Modifications to the constant regioncompatible with this invention comprise additions, deletions orsubstitutions of one or more amino acids in one or more domains. Thatis, the modified antibodies disclosed herein may comprise alterations ormodifications to one or more of the three heavy chain constant domains(CH1, CH2 or CH3) and/or to the light chain constant domain (CL). Insome embodiments of the invention modified constant regions wherein oneor more domains are partially or entirely deleted are contemplated. Inother embodiments the modified antibodies will comprise domain deletedconstructs or variants wherein the entire CH2 domain has been removed(ΔCH2 constructs). In still other embodiments the omitted constantregion domain will be replaced by a short amino acid spacer (e.g. 10residues) that provides some of the molecular flexibility typicallyimparted by the absent constant region.

Besides their configuration, it is known in the art that the constantregion mediates several effector functions. For example, binding of theC1 component of complement to antibodies activates the complementsystem. Activation of complement is important in the opsonization andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and can also be involved in autoimmunehypersensitivity. Further, antibodies bind to cells via the Fc region,with a Fc receptor site on the antibody Fc region binding to a Fcreceptor (FcR) on a cell. There are a number of Fc receptors which arespecific for different classes of antibody, including IgG (gammareceptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mureceptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), release of inflammatory mediators, placental transfer and controlof immunoglobulin production. Although various Fc receptors and receptorsites have been studied to a certain extent, there is still much whichis unknown about their location, structure and functioning.

While not limiting the scope of the present invention, it is believedthat antibodies comprising constant regions modified as described hereinprovide for altered effector functions that, in turn, affect thebiological profile of the administered antibody. For example, thedeletion or inactivation (through point mutations or other means) of aconstant region domain may reduce Fc receptor binding of the circulatingmodified antibody thereby increasing tumor localization. In other casesit may be that constant region modifications, consistent with thisinvention, moderate complement binding and thus reduce the serum halflife and nonspecific association of a conjugated cytotoxin. Yet othermodifications of the constant region may be used to eliminate disulfidelinkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. Similarly, modifications to the constant region inaccordance with this invention may easily be made using well knownbiochemical or molecular engineering techniques.

It will be noted that the modified antibodies may be engineered to fusethe CH3 domain directly to the hinge region of the respective modifiedantibodies. In other constructs it may be desirable to provide a peptidespacer between the hinge region and the modified CH2 and/or CH3 domains.For example, compatible constructs could be expressed wherein the CH2domain has been deleted and the remaining CH3 domain (modified orunmodified) is joined to the hinge region with a 5-20 amino acid spacer.Such a spacer may be added, for instance, to ensure that the regulatoryelements of the constant domain remain free and accessible or that thehinge region remains flexible. However, it should be noted that aminoacid spacers may, in some cases, prove to be immunogenic and elicit anunwanted immune response against the construct. Accordingly, any spaceradded to the construct be relatively non-immunogenic or, even omittedaltogether if the desired biochemical qualities of the modifiedantibodies may be maintained.

Besides the deletion of whole constant region domains, it will beappreciated that the antibodies of the present invention may be providedby the partial deletion or substitution of a few or even a single aminoacid. For example, the mutation of a single amino acid in selected areasof the CH2 domain may be enough to substantially reduce Fc binding andthereby increase tumor localization. Similarly, it may be desirable tosimply delete that part of one or more constant region domains thatcontrol the effector function (e.g. complement CLQ binding) to bemodulated. Such partial deletions of the constant regions may improveselected characteristics of the antibody (serum half-life) while leavingother desirable functions associated with the subject constant regiondomain intact. Moreover, as alluded to above, the constant regions ofthe disclosed antibodies may be modified through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it may be possible to disrupt theactivity provided by a conserved binding site (e.g. Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Yet other embodiments may comprise the additionof one or more amino acids to the constant region to enhance desirablecharacteristics such as effector function or provide for more cytotoxinor carbohydrate attachment. In such embodiments it can be desirable toinsert or replicate specific sequences derived from selected constantregion domains.

In certain embodiments of the invention, it can be desirable to use anantibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. Various techniques are known for theproduction of antibody fragments. Traditionally, these fragments arederived via proteolytic digestion of intact antibodies (for exampleMorimoto et al., 1993, Journal of Biochemical and Biophysical Methods24:107-117 and Brennan et al., 1985, Science, 229:81). However, thesefragments are now typically produced directly by recombinant host cellsas described above. Thus Fab, Fv, and scFv antibody fragments can all beexpressed in, and secreted from, E. coli or other host cells, thusallowing the production of large amounts of these fragments.Alternatively, such antibody fragments can be isolated from the antibodyphage libraries discussed herein. The antibody fragment can also belinear antibodies as described in U.S. Pat. No. 5,641,870, for example,and can be monospecific or bispecific. Other techniques for theproduction of antibody fragments will be apparent to one of skill in theart.

It can further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e. thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art.

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent. Cytotoxic agents includechemotherapeutic agents, growth inhibitory agents, toxins (e.g., anenzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof), radioactive isotopes (i.e., aradioconjugate), etc. Chemotherapeutic agents useful in the generationof such immunoconjugates include, for example, methotrexate, adriamicin,doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents. Enzymatically active toxins and fragments thereofthat can be used include diphtheria A chain, nonbinding active fragmentsof diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthinproteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, andthe tricothecenes. In some embodiments, the antibodies can be conjugatedto radioisotopes, such as ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹²³I, ¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm,⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re and ¹⁸⁸Re using anyone of a number ofwell known chelators or direct labeling. In other embodiments, thedisclosed compositions can comprise antibodies coupled to drugs,prodrugs, or lymphokines such as interferon. Conjugates of the antibodyand cytotoxic agent are made using a variety of bifunctionalprotein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azidocompounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as tolyene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Conjugatesof an antibody and one or more small molecule toxins, such as acalicheamicin, maytansinoids, a trichothene, and CC 1065, and thederivatives of these toxins that have toxin activity, can also be used.In some embodiments, the modified antibodies can be complexed with otherimmunologically active ligands (e.g., antibodies or fragments thereof)wherein the resulting molecule binds to both the neoplastic cell and aneffector cell such as a T cell.

Regardless of how useful quantities are obtained, the antibodies of thepresent invention can be used in any one of a number of conjugated (i.e.an immunoconjugate) or unconjugated forms. Alternatively, the antibodiesof this invention can be used in a nonconjugated or “naked” form toharness the subject's natural defense mechanisms includingcomplement-dependent cytotoxicity (CDC) and antibody dependent cellulartoxicity (ADCC) to eliminate the malignant cells. The selection of whichconjugated or unconjugated modified antibody to use will depend of thetype and stage of cancer, use of adjunct treatment (e.g., chemotherapyor external radiation) and patient condition. It will be appreciatedthat one could readily make such a selection in view of the teachingsherein.

Competition assays can be used to determine whether two antibodies bindthe same epitope by recognizing identical or sterically overlappingepitopes. Any method known to one of skill in the art for determiningcompetitive binding (such as e.g., the immunoassays described elsewhereherein) may be used.

The antibodies of the present invention can be assayed forimmunospecific binding by any method known in the art. The immunoassayswhich can be used include, but are not limited to, competitive andnon-competitive assay systems using techniques such as Biacore analysis,FACS analysis, immunofluorescence, immunocytochemistry, Western blotanalysis, radioimmunoassay, ELISA, “sandwich” immunoassay,immunoprecipitation assay, precipitin reaction, gel diffusion precipitinreaction, immunodiffusion assay, agglutination assay,complement-fixation assay, immunoradiometric assay, fluorescentimmunoassay, and protein A immunoassay. Such assays are routine and wellknown in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocolsin Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, whichis incorporated by reference herein in its entirety).

In some embodiments, of the present invention the immunospecificity ofan antibody against a membrane proximal region of the extracellulardomain of a human Notch1 receptor is determined using ELISA. An ELISAassay comprises preparing antigen, coating wells of a 96 well microtiterplate with antigen, adding the antibody against a cancer stem cellmarker conjugated to a detectable compound such as an enzymaticsubstrate (e.g. horseradish peroxidase or alkaline phosphatase) to thewell, incubating for a period of time and detecting the presence of theantigen. Alternatively the antibody against a membrane proximal regionof the extracellular domain of a human Notch1 receptor is not conjugatedto a detectable compound, but instead a second conjugated antibody thatrecognizes the antibody against a membrane proximal region of theextracellular domain of a human Notch1 receptor is added to the well.Further, instead of coating the well with the antigen, the antibodyagainst a membrane proximal region of the extracellular domain of ahuman Notch1 receptor can be coated to the well and a second antibodyconjugated to a detectable compound can be added following the additionof the antigen to the coated well. It is known to one of skill in theart what parameters can be modified to increase the signal detected aswell as other variations of ELISAs known in the art (see e.g. Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 11.2.1).

The binding affinity of an antibody to a membrane proximal region of theextracellular domain of Notch1 receptor and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g. ³H or ¹²⁵I), orfragment or variant thereof, with the antibody of interest in thepresence of increasing amounts of unlabeled antigen followed by thedetection of the antibody bound to the labeled antigen. The affinity ofthe antibody against a membrane proximal region of the extracellulardomain of a human Notch1 receptor and the binding off-rates can bedetermined from the data by Scatchard plot analysis. In someembodiments, Biacore kinetic analysis is used to determine the bindingon and off rates of antibodies against a membrane proximal region of theextracellular domain of a human Notch1 receptor. Biacore kineticanalysis comprises analyzing the binding and dissociation of antibodiesfrom chips with immobilized antigen, for example, Notch1 receptors, ontheir surface.

In certain embodiments, the invention encompasses isolatedpolynucleotides that encode a polypeptide comprising an antibody orfragment thereof, against a non-ligand binding membrane proximal regionof the extracellular domain of a human Notch1 receptor. The term“polynucleotide encoding a polypeptide” encompasses a polynucleotidewhich includes only coding sequences for the polypeptide as well as apolynucleotide which includes additional coding and/or non-codingsequences. The polynucleotides of the invention can be in the form ofRNA or in the form of DNA. DNA includes cDNA, genomic DNA, and syntheticDNA; and can be double-stranded or single-stranded, and if singlestranded can be the coding strand or non-coding (anti-sense) strand. Thepolynucleotides of the invention can be in the form of RNA or in theform of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.The DNA can be double-stranded or single-stranded, and if singlestranded can be the coding strand or non-coding (anti-sense) strand.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs, andderivatives. The variant of the polynucleotide can be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide.

As hereinabove indicated, the polynucleotide can have a coding sequencewhich is a naturally occurring allelic variant of the coding sequence ofthe disclosed polypeptides. As known in the art, an allelic variant isan alternate form of a polynucleotide sequence which has a substitution,deletion or addition of one or more nucleotides, which does notsubstantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide can be fused in the same readingframe to a polynucleotide which aids in expression and secretion of apolypeptide from a host cell, for example, a leader sequence whichfunctions as a secretory sequence for controlling transport of apolypeptide from the cell. The polypeptide having a leader sequence is apreprotein and can have the leader sequence cleaved by the host cell toform the mature form of the polypeptide. The polynucleotides can alsoencode for a proprotein which is the mature protein plus additional 5′amino acid residues. A mature protein having a prosequence is aproprotein and is an inactive form of the protein. Once the prosequenceis cleaved an active mature protein remains. Thus, for example, thepolynucleotide of the present invention can encode for a mature protein,or for a protein having a prosequence or for a protein having both aprosequence and presequence (leader sequence).

The polynucleotides of the present invention can also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence can be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencecan be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson et al., 1984, Cell 37:767).

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 90% identical, 95% identical, and in some embodiments, at least96%, 97%, 98% or 99% identical to the disclosed sequences. In someembodiments, the polynucleotides have a nucleotide sequence at least 90%identical to SEQ ID NOs: 3, 5, 7, 9, 11, 13, 21, 25 or 29 (with orwithout signal sequence). In some embodiments, the polynucleotides havea nucleotide sequence at least 90% identical to SEQ ID NOs:7 or 13. Insome embodiments, the invention provides a polynucleotide thathybridizes to a polynucleotide encoding the polypeptides of SEQ IDNOs:4, 6, 8, 10, 12, 14, 22, 23, 24, 26, 27, 28, 30, 31, or 32. In someembodiments, the polynucleotides hybridize to the polynucleotides of SEQID NOs:3, 5, 7, 9, 11, 13, 21, 25 or 29. In some embodiments, thepolynucleotides hybridize under stringent hybridization conditions.

As used herein, the phrases “hybridizes” or “selectively hybridizes” or“specifically hybridizes” refer to the binding or duplexing of amolecule only to a particular nucleotide sequence under stringenthybridization conditions when that sequence is present in a complexmixture (e.g., a library of DNAs or RNAs). See, e.g., Andersen (1998)Nucleic Acid Hybridization Springer-Verlag; Ross (ed. 1997) Nucleic AcidHybridization Wiley.

As used herein, the phrase “stringent hybridization conditions” refersto conditions under which a probe will hybridize to its targetsubsequence, typically in a complex mixture of nucleic acid, but to noother sequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength. The Tm is the temperature(under defined ionic strength, pH, and nucleic concentration) at which50% of the probes complementary to the target hybridize to the targetsequence at equilibrium (as the target sequences are present in excess,at Tm, 50% of the probes are occupied at equilibrium). Stringentconditions will be those in which the salt concentration is less thanabout 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ionconcentration (or other salts) at pH 7.0 to 8.3 and the temperature isat least about 30° C. for short probes (e.g., 10 to 50 nucleotides) andat least about 60° C. for long probes (e.g., greater than 50nucleotides). Stringent conditions can also be achieved with theaddition of destabilizing agents such as formamide. For high stringencyhybridization, a positive signal is at least two times background, or 10times background hybridization. Exemplary high stringency or stringenthybridization conditions include: 50% formamide, 5×SSC, and 1% SDSincubated at 42° C. or 5×SSC and 1% SDS incubated at 65° C., with a washin 0.2×SSC and 0.1% SDS at 65° C. For PCR, a temperature of about 36° C.is typical for low stringency amplification, although annealingtemperatures can vary from about 32° C. to about 48° C. depending onprimer length. For high stringency PCR amplification, a temperature ofabout 62° C. is typical, although high stringency annealing temperaturescan range from about 50° C. to about 65° C., depending on the primerlength and specificity. Typical cycle conditions for both high and lowstringency amplifications include a denaturation phase of 90° C. to 95°C. for 30-120 sec, an annealing phase lasting 30-120 sec, and anextension phase of about 72° C. for 1-2 min.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence can include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence can be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted intothe reference sequence. These mutations of the reference sequence canoccur at the amino- or carboxy-terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence.

As a practical matter, whether any particular nucleic acid molecule isat least 95%, 96%, 97%, 98% or 99% identical to a reference sequence canbe determined conventionally using known computer programs such as theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). Bestfit uses the local homology algorithmof Smith and Waterman, Advances in Applied Mathematics 2: 482 489(1981), to find the best segment of homology between two sequences. Whenusing Bestfit or any other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, such that the percentage of identity is calculatedover the full length of the reference nucleotide sequence and that gapsin homology of up to 5% of the total number of nucleotides in thereference sequence are allowed.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In some embodiments,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those preferred by abacterial host such as E. coli).

The polypeptides of the present invention can be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides comprisingan antibody, or fragment thereof, against a non-ligand binding membraneproximal region of the extracellular domain of a human Notch1 receptor.It will be recognized in the art that some amino acid sequences of theinvention can be varied without significant effect of the structure orfunction of the protein. Thus, the invention further includes variationsof the polypeptides which show substantial activity or which includeregions of an antibody, or fragment thereof, against a membrane proximalregion of the extracellular domain of a human Notch1 receptor. Suchmutants include deletions, insertions, inversions, repeats, and typesubstitutions.

The polypeptides and polynucleotides of the present invention areprovided in an isolated form, and at times are purified to homogeneity.

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthesis methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host. For example, cDNA can be obtained by screening a humancDNA library with a labeled DNA fragment encoding a polypeptide (forexample, nucleotide SEQ ID NO:1) and identifying positive clones byautoradiography. Further rounds of plaque purification and hybridizationare performed using conventional methods.

In some embodiments of a recombinant method, a DNA sequence isconstructed by isolating or synthesizing a DNA sequence encoding awild-type protein of interest. Optionally, the sequence can bemutagenized by site-specific mutagenesis to provide functional analogsthereof. (See, e.g. Zoeller et al., 1984, Proc.-Nat. Acad. Sci. USA,81:5662-5066 and U.S. Pat. No. 4,588,585.) Another method ofconstructing a DNA sequence encoding a polypeptide of interest would beby chemical synthesis using an oligonucleotide synthesizer. Sucholigonucleotides can be designed based on the amino acid sequence of thedesired polypeptide and selecting those codons that are favored in thehost cell in which the recombinant polypeptide of interest will beproduced.

Standard methods can be applied to synthesize an isolated polynucleotidesequence encoding an isolated polypeptide of interest. For example, acomplete amino acid sequence can be used to construct a back-translatedgene. Further, a DNA oligomer containing a nucleotide sequence codingfor the particular isolated polypeptide can be synthesized. For example,several small oligonucleotides coding for portions of the desiredpolypeptide can be synthesized and then ligated. The individualoligonucleotides typically contain 5′ or 3′ overhangs for complementaryassembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the mutant DNA sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene is operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

Recombinant expression vectors are used to amplify and express DNAencoding cancer stem cell marker polypeptide fusions. Recombinantexpression vectors are replicable DNA constructs which have synthetic orcDNA-derived DNA fragments encoding a cancer stem cell markerpolypeptide fusion or a bioequivalent analog operatively linked tosuitable transcriptional or translational regulatory elements derivedfrom mammalian, microbial, viral or insect genes. A transcriptional unitgenerally comprises an assembly of (1) a genetic element or elementshaving a regulatory role in gene expression, for example,transcriptional promoters or enhancers, (2) a structural or codingsequence which is transcribed into mRNA and translated into protein, and(3) appropriate transcription and translation initiation and terminationsequences, as described in detail below. Such regulatory elements caninclude an operator sequence to control transcription. The ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants canadditionally be incorporated. DNA regions are operatively linked whenthey are functionally related to each other. For example, DNA for asignal peptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Generally, operatively linkedmeans contiguous and, in the case of secretory leaders, means contiguousand in reading frame. Structural elements intended for use in yeastexpression systems include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, whererecombinant protein is expressed without a leader or transport sequence,it can include an N-terminal methionine residue. This residue canoptionally be subsequently cleaved from the expressed recombinantprotein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Esherichia coli,including pCR1, pBR322, pMB9 and their derivatives, and wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a cancer stem cell marker proteininclude prokaryotes, yeast, insect or higher eukaryotic cells.Prokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. Higher eukaryotic cells include establishedcell lines of mammalian origin as described below. Cell-free translationsystems could also be employed. Appropriate cloning and expressionvectors for use with bacterial, fungal, yeast, and mammalian cellularhosts are described by Pouwels et al. (Cloning Vectors: A LaboratoryManual, Elsevier, N.Y., 1985), the relevant disclosure of which ishereby incorporated by reference.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (1981, Cell,23:175), and other cell lines capable of expressing an appropriatevector including, for example, L cells, C127, 3T3, Chinese hamster ovary(CHO), HeLa and BHK cell lines. Mammalian expression vectors cancomprise nontranscribed elements such as an origin of replication, asuitable promoter and enhancer linked to the gene to be expressed, andother 5′ or 3′ flanking nontranscribed sequences, and 5′ or 3′nontranslated sequences, such as necessary ribosome binding sites, apolyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences. Baculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, 1988, Bio/Technology, 6:47.

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be physically characterized using such techniques asproteolysis, nuclear magnetic resonance and x-ray crystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a recombinant protein or cancer stem cellprotein-Fc composition. Some or all of the foregoing purification steps,in various combinations, can also be employed to provide a homogeneousrecombinant protein.

Recombinant protein produced in bacterial culture is usually isolated byinitial extraction from cell pellets, followed by one or moreconcentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

The present invention also provides methods for inhibiting the growth oftumorigenic cells expressing a cancer stem cell marker using theantagonists of a cancer stem cell marker described herein. In someembodiments, the method of inhibiting the growth of tumorigenic cellsexpressing a cancer stem cell marker, for example Notch1 receptor,comprises contacting the cell with an antagonist against a cancer stemcell marker in vitro. For example, an immortalized cell line or a cancercell line that expresses a cancer stem cell marker is cultured in mediumto which is added an antagonist of the expressed cancer stem cell markerto inhibit cell growth. In some embodiments, tumor cells comprisingtumor stem cells are isolated from a patient sample such as, forexample, a tissue biopsy, pleural effusion, or blood sample and culturedin medium to which is added an antagonist of a cancer stem cell markerto inhibit cell growth. In some embodiments, the antagonist is anantibody that specifically recognizes an epitope of a cancer stem cellmarker protein. For example, antibodies against a cancer stem cellmarker protein can be added to the culture medium of isolated cancerstem cells to inhibit cell growth.

In some embodiments, the method of inhibiting the growth of tumorigeniccells expressing a cancer stem cell marker comprises contacting the cellwith an antagonist against a cancer stem cell marker in vivo. In someembodiments, the method of inhibiting growth of tumorigenic cellsexpressing Notch1 comprises contacting the cells with an antibody thatspecifically binds to a non-ligand binding membrane proximal region of ahuman Notch1 receptor. In some embodiments, the antibody inhibits growthof tumorigenic cells by inhibiting the activity of Notch1. In someembodiments, the antibody inhibits growth of tumorigenic cells byinhibiting ligand-induced Notch1 signaling. In some embodiments, theantibody inhibits growth of tumorigenic cells by inhibiting the cleavageof Notch1. In some embodiments, the antibody inhibits growth oftumorigenic cells by reducing the frequency or the number of cancer stemcells in the tumor.

In certain embodiments, contacting a tumorigenic cell with an antagonistto a cancer stem cell marker is undertaken in an animal model. Forexample, xenografts expressing a cancer stem cell marker are grown inimmunocompromised mice (e.g. NOD/SCID mice) that are administered anantagonist to a cancer stem cell marker to inhibit tumor growth. In someembodiments, cancer stem cells that express a cancer stem cell markerare isolated from a patient sample such as, for example, a tissuebiopsy, pleural effusion, or blood sample and injected intoimmunocompromised mice that are then administered an antagonist againstthe cancer stem cell marker to inhibit tumor cell growth. In someembodiments, the antagonist of a cancer stem cell marker is administeredat the same time or shortly after introduction of tumorigenic cells intothe animal to prevent tumor growth. In other embodiments, the antibodyagainst the cancer stem cell marker is administered as a therapeuticagent after the tumorigenic cells have grown to a specified size.

The present invention further provides pharmaceutical compositionscomprising antibodies, polypeptides or other agents that target a cancerstem cell marker. These pharmaceutical compositions find use ininhibiting tumor growth, tumor cell growth and treating cancer in humanpatients.

Formulations are prepared for storage and use by combining a purifiedantagonist (e.g., antibody) of the present invention with apharmaceutically acceptable vehicle (e.g., carrier, excipient, etc.)(Remington, The Science and Practice of Pharmacy 20th Edition MackPublishing, 2000). Suitable pharmaceutically acceptable vehiclesinclude, but are not limited to, nontoxic buffers such as phosphate,citrate, and other organic acids; salts such as sodium chloride;antioxidants including ascorbic acid and methionine; preservatives suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weightpolypeptides (less than about 10 amino acid residues); proteins such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; carbohydrates such asmonosacchandes, disaccharides, glucose, mannose, or dextrins; chelatingagents such as EDTA; sugars such as sucrose, mannitol, trehalose orsorbitol; salt-forming counter-ions such as sodium; metal complexes suchas Zn-protein complexes; and/or non-ionic surfactants such as TWEEN orpolyethylene glycol (PEG).

The pharmaceutical composition of the present invention can beadministered in any number of ways for either local or systemictreatment. Administration can be topical (such as to mucous membranesincluding vaginal and rectal delivery) such as transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders; pulmonary such as by inhalation or insufflation of powdersor aerosols (including by nebulizer), intratracheal, intranasal,epidermal and transdermal; oral; parenteral including intravenous,intraarterial, intratumoral, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial such asintrathecalor intraventricular.

The therapeutic formulation can be in unit dosage form. Suchformulations include tablets, pills, capsules, powders, granules,solutions or suspensions in water or non-aqueous media, or suppositoriesfor oral, parenteral, or rectal administration or for administration byinhalation. In solid compositions such as tablets the principal activeingredient is mixed with a pharmaceutical carrier. Conventionaltableting ingredients include corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother diluents (e.g., water) to form a solid preformulation compositioncontaining a homogeneous mixture of a compound of the present invention,or a non-toxic pharmaceutically acceptable salt thereof. The solidpreformulation composition is then subdivided into unit dosage forms ofthe type described herein. The tablets, pills, etc of the novelcomposition can be coated or otherwise compounded to provide a dosageform affording the advantage of prolonged action. For example, thetablet or pill can comprise an inner composition covered by an outercomponent. Furthermore, the two components can be separated by anenteric layer that serves to resist disintegration and permits the innercomponent to pass intact through the stomach or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

Pharmaceutical formulations include antibodies of the present inventioncomplexed with liposomes (Epstein, et al., 1985, Proc. Natl. Acad. Sci.USA, 82:3688; Hwang, et al., 1980, Proc. Natl. Acad. Sci. USA, 77:4030;and U.S. Pat. Nos. 4,485,045 and 4,544,545). Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556. Someliposomes can be generated by the reverse phase evaporation with a lipidcomposition comprising phosphatidylcholine, cholesterol, andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter.

The antibodies can also be entrapped in microcapsules. Suchmicrocapsules are prepared, for example, by coacervation techniques orby interfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions as described in Remington, TheScience and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

In addition sustained-release preparations can be prepared. Suitableexamples of sustained-release preparations include semi-permeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles (e.g. films, ormicrocapsules). Examples of sustained-release matrices includepolyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) orpoly(v nylalcohol), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and 7 ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), sucrose acetateisobutyrate, and poly-D(−)-3-hydroxybutyric acid. In some embodimentsthe antibodies can be used to treat various conditions characterized byexpression and/or increased responsiveness of cells to a cancer stemcell marker. Particularly it is envisioned that the antibodies against acancer stem cell marker, for example Notch1, will be used to treatproliferative disorders including but not limited to benign andmalignant tumors of the kidney, liver, bladder, breast, stomach, ovary,colon, rectum, prostate, lung, vulva, thyroid, head and neck, brain(glioblastoma, astrocytoma, medulloblastoma, etc), blood and lymph(leukemias and lymphomas).

In some embodiments, the treatment involves the combined administrationof an antibody or other agent of the present invention and achemotherapeutic agent or cocktail of multiple differentchemotherapeutic agents. Treatment with an antibody can occur prior to,concurrently with, or subsequent to administration of chemotherapies.Chemotherapies contemplated by the invention include chemical substancesor drugs which are known in the art and are commercially available, suchas doxorubicin, 5-fluorouracil, cytosine arabinoside (“Ara-C”),cyclophosphamide, thiotepa, busulfan, cytoxin, taxol, methotrexate,cisplatin, melphalan, vinblastine and carboplatin. Combinedadministration can include co-administration, either in a singlepharmaceutical formulation or using separate formulations, orconsecutive administration in either order but generally within a timeperiod such that all active agents can exert their biological activitiessimultaneously. Preparation and dosing schedules for suchchemotherapeutic agents can be used according to manufacturers'instructions or as determined empirically. Preparation and dosingschedules for such chemotherapy are also described in ChemotherapyService Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992).

Chemotherapeutic agents useful in the instant invention also include,but are not limited to, alkylating agents such as thiotepa andcyclophosphamide (Cytoxan); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime nitrogen mustardssuch as chlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (Taxotere, Rhone-Poulenc Rorer, Antony, France); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Chemotherapeutic agents also includeanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);and antiandrogens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

In certain embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerase inhibitors are chemotherapy agents thatinterfere with the action of a topoisomerase enzyme (e.g., topoisomeraseI or II). Topoisomerase inhibitors include, but are not limited to,doxorubicin HCL, daunorubicin citrate, mitoxantrone HCL, actinomycin D,etoposide, topotecan HCL, teniposide (VM-26), and irinotecan.

In certain embodiments, the chemotherapeutic agent is ananti-metabolite. An anti-metabolite is a chemical with a structure thatis similar to a metabolite required for normal biochemical reactions,yet different enough to interfere with one or more normal functions ofcells, such as cell division. Anti-metabolites include, but are notlimited to, gemcitabine, fluorouracil, capecitabine, methotrexatesodium, ralitrexed, Pemetrexed, tegafur, cytosine arabinoside,Thioguanine (GlaxoSmithKline), 5-azacytidine, 6-mercaptopurine,azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, andcladribine, as well as pharmaceutically acceptable salts, acids, orderivatives of any of these.

In other embodiments, the treatment involves the combined administrationof an antibody or other agent of the present invention and radiationtherapy. Treatment with an antibody can occur prior to, concurrentlywith, or subsequent to administration of radiation therapy. Any dosingschedules for such radiation therapy can be used as determined by theskilled practitioner.

In other embodiments, the treatment can involve the combinedadministration of antibodies of the present invention with otherantibodies against additional tumor associated antigens including, butnot limited to, antibodies that bind to the EGF receptor (EGFR)(Erbitux®), the erbB2 receptor (HER2) (Herceptin®), and vascularendothelial growth factor (VEGF) (Avastin®). Furthermore, treatment caninclude administration of one or more cytokines; can be accompanied bysurgical removal of cancer cells; and/or any other therapy deemednecessary by a treating physician.

For the treatment of the disease, the appropriate dosage of an antibodyor other agent of the present invention depends on the type of diseaseto be treated, the severity and course of the disease, theresponsiveness of the disease, whether the antibody is administered fortherapeutic or preventative purposes, previous therapy, patient'sclinical history, and so on all at the discretion of the treatingphysician. The antibody or agent can be administered one time or over aseries of treatments lasting from several days to several months, oruntil a cure is effected or a diminution of the disease state isachieved (e.g. reduction in tumor size). Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient and will vary depending on the relative potency of an individualantagonist. The administering physician can easily determine optimumdosages, dosing methodologies and repetition rates. In general, dosageis from 0.01 μg to 100 mg per kg of body weight, and can be given onceor more daily, weekly, monthly or yearly. The treating physician canestimate repetition rates for dosing based on measured residence timesand concentrations of the antibody or agent in bodily fluids or tissues.

The present invention provides kits comprising the antibodies describedherein and that can be used to perform the methods described herein. Insome embodiments, a kit comprises at least one purified antibody againsta cancer stem cell marker, in one or more containers. In someembodiments, a kit comprises at least one purified antibody against anon-ligand binding membrane proximal region of the extracellular domainof a human Notch1 receptor, in one or more containers. In someembodiments, a kit comprises the antibody 52M51 or a humanized variantof 52M51. In some embodiments, a kit comprises the antibody 52R43. Insome embodiments, the kits contain all of the components necessaryand/or sufficient to perform a detection assay, including all controls,directions for performing assays, and any necessary software foranalysis and presentation of results. One skilled in the art willreadily recognize that the disclosed antibodies of the present inventioncan be readily incorporated into one of the established kit formatswhich are well known in the art.

In certain embodiments, the present invention provides a method ofidentifying a molecule that binds to a non-ligand binding membraneproximal region of the extracellular domain of a human Notch1 receptorand inhibits tumor growth, the method comprising: i) incubating themolecule with the non-ligand binding membrane proximal region of theextracellular domain of the human Notch1 receptor; ii) determining ifthe molecule binds to the membrane proximal region of the extracellulardomain of the human Notch receptor; and iii) determining if the moleculeinhibits tumor growth. Molecules that specifically bind a membraneproximal region of the extracellular domain of a human Notch1 receptorinclude, but are not limited to, polypeptides and antibodies.

Screening can be performed using any suitable method known in the art.In certain embodiments, screening is performed in vitro. In someembodiments, cells expressing a non-ligand binding membrane proximalregion of the extracellular domain of a human Notch1 receptor areincubated with a labeled molecule and specific binding of the labeledmolecule to a membrane proximal region of the extracellular domain of ahuman Notch1 receptor is determined by FACS analysis. In someembodiments, a non-ligand binding membrane proximal region of theextracellular domain of a human Notch1 receptor is expressed by phagedisplay, and molecules that specifically binding to a membrane proximalregion of the extracellular domain of a human Notch1 receptor areidentified. Other suitable methods for identifying molecules thatspecifically bind to a non-ligand binding membrane proximal region of ahuman Notch1 receptor include, but are not limited to, ELISA; Western(or immuno) blotting; and yeast-two-hybrid.

Molecules that specifically bind to a non-ligand binding membraneproximal region of the extracellular domain of a human Notch1 receptorare then tested for inhibition of tumor cell growth. Testing can beperformed using any suitable method known in the art. In certainembodiments, molecules that specifically bind to membrane proximalregion of the extracellular domain of a human Notch1 receptor are testedfor the ability to inhibit tumor growth in vitro. In some embodiments,molecules that specifically bind a membrane proximal region of theextracellular domain of a human Notch1 receptor are incubated with tumorcells in culture and proliferation of tumor cells in the presence of themolecule that specifically binds a membrane proximal region of theextracellular domain of a human Notch1 receptor is determined andcompared to tumor cells incubated with a non-binding control molecule.In certain embodiments, molecules that specifically bind to non-ligandbinding membrane proximal region of the extracellular domain of a humanNotch1 receptor are tested for the ability to inhibit tumor growth invivo. In certain embodiments, molecules that specifically bind amembrane proximal region of the extracellular domain of a human Notch1receptor are injected into an animal xenograft model and the growth oftumors in animals treated with molecules that specifically bind to themembrane proximal region of the extracellular domain of a human Notch1receptor is determined and compared to animals treated with anon-binding control molecule.

EXAMPLES Example 1

Antibodies were generated against a non-ligand binding region of Notch1,specifically the non-ligand binding membrane proximal region of theextracellular domain. In certain embodiments, recombinant polypeptidefragments of the human Notch1 extracellular domain were generated asantigens for antibody production. Standard recombinant DNA technologywas used to isolate polynucleotides encoding the membrane proximalregion of the extracellular domain of human Notch1 amino acids 1427-1732(SEQ ID NO:1). These polynucleotides were separately ligated in-frameN-terminal to a human Fc and histidine-tag and cloned into a transferplasmid vector for baculovirus-mediated expression in insect cells.Standard transfection, infection, and cell culture protocols were usedto produce recombinant insect cells expressing the corresponding Notch1polypeptide corresponding to a membrane proximal region comprising aminoacids 1427-1732 (SEQ ID NO:2) (O'Reilly et al., 1994, BaculovirusExpression Vectors: A Laboratory Manual, Oxford: Oxford UniversityPress).

Notch1 membrane proximal region (Notch1 amino acids 1472-1732)polypeptide was purified from insect cell lysates using protein A andNi++-chelate affinity chromatography as known to one skilled in the art.Purified Notch1 membrane proximal region polypeptide was dialyzedagainst PBS (pH=7), concentrated to approximately 1 mg/ml, and sterilefiltered in preparation for immunization.

Mice (n=3) were immunized with purified Notch1 antigen protein (AntibodySolutions; Mountain View, Calif.) using standard techniques. Blood fromindividual mice was screened approximately 70 days after initialimmunization for antigen recognition using ELISA and FACS analysis (asdescribed herein). The two animals with the highest antibody titers wereselected for final antigen boost after which spleen cells were isolatedfor hybridoma production. Hybridoma cells were plated at 1 cell per wellin 96 well plates, and the supernatant from each well screened by ELISAand FACS analysis against Notch1 membrane proximal region polypeptide.Several hybridomas with high antibody titer were selected and scaled upin static flask culture. Antibodies were purified from the hybridomasupernatant using protein A or protein G agarose chromatography.Purified monoclonal antibodies were tested again by FACS as describedherein. Several antibodies that recognized the membrane proximal regionof the extracellular domain of human Notch1 were isolated. A hydridomacell line expressing antibody 52M51 was deposited with ATCC under theconditions of the Budapest Treaty on Aug. 7, 2008 and assigned ATTCPatent Deposit Designation PTA-9405. The nucleotide and predictedprotein sequences of both the heavy chain (SEQ ID NO:9 and 10) and lightchain (SEQ ID NO:3 and 4) of antibody 52M51 were determined.

Human Antibodies

In alternative embodiments, human antibodies that specifically recognizethe non-ligand binding membrane proximal region of the extracellulardomain of a Notch1 receptor are isolated using phage display technology.In certain embodiments, a synthetic antibody library containing humanantibody variable domains is screened for specific and high affinityrecognition of a Notch receptor antigen described herein. In certainembodiments, a human Fab phage display library is screened using aseries of recombinant proteins comprising the non-ligand bindingmembrane proximal region of the extracellular domain of a Notch 1receptor. Briefly, 2×10¹³ Fab displaying phage particles are incubatedwith recombinant protein (passively immobilized) in round one, thenon-specific phage are washed off, and then specific phage are elutedwith either low pH (cells) or DTT (recombinant protein). The elutedoutput is used to infect TG1 F+ bacteria, rescued with helper phage, andthen Fab display induced with IPTG (0.25 mM). This process is repeatedfor two additional rounds and then round three is screened in ELISAagainst passively immobilized antigen (5 μg/ml).

CDR cassettes in the library are specifically exchanged via uniqueflanking restriction sites for antibody optimization. Optimized humanvariable regions are then cloned into an Ig expression vector containinghuman IgG1 heavy-chain and kappa light-chain for expression of humanantibodies in CHO cells.

Epitope Mapping

To identify antibodies that recognize specific a non-ligand bindingmembrane proximal region of the Notch1 receptor extracellular domains,epitope mapping is performed. In certain embodiments, mammalianexpression plasmid vectors comprising a CMV promoter upstream ofpolynucleotides that encode fragments of the extracellular Notch1 domainas Fc fusion proteins are generated using standard recombinant DNAtechnology. In certain embodiments, epitope mapping of the 52M series ofnon-ligand binding region antibodies is done using a series of fusionproteins and deletions of the membrane proximal region of theextracellular domain of a human Notch1 from about amino acid 1427 toabout amino acid 1732. These recombinant fusion proteins are expressedin transiently transfected HEK 293 cells from which conditioned mediumis collected twenty-four to forty-eight hours post-transfection forELISA.

In certain embodiments, the Notch1 fusion protein fragments areseparated on SDS-PAGE gels and probed with both anti-Fc antibodies todetect the presence of all fusion proteins versus anti-Notch1 antibodiesto detect the domains recognized by each anti-Notch antibody.

To identify specific epitopes within the extracellular domainsrecognized by an antibody against Notch1 the SPOTs system is used (SigmaGenosys, The Woodlands, Tex.). A series of 10-residue linear peptidesoverlapping by one amino acid and covering the entire Notch1extracellular domain are synthesized and covalently bound to a cellulosemembrane by the SPOT synthesis technique. The membrane is preincubatedfor 8 hours at room temperature with blocking buffer and hybridized withantibody overnight at 4° C. The membrane is then washed, incubated witha secondary antibody conjugated to horseradish peroxidase (HRP)(Amersham Bioscience, Piscataway, N.J.), re-washed, and visualized withsignal development solution containing 3-amino-9-ethylcarbazole.Specific epitopes recognized by an antibody are thus determined.

Chimeric Antibodies

After monoclonal antibodies that specifically recognize a non-ligandbinding membrane proximal domain of the extracellular domain of a Notch1receptor are identified, these antibodies are modified to overcome thehuman anti-mouse antibody (HAMA) immune response when rodent antibodiesare used as therapeutics agents. The variable regions of the heavy-chainand light-chain of the selected monoclonal antibody are isolated byRT-PCR from hybridoma cells and ligated in-frame to human IgG1heavy-chain and kappa light chain constant regions, respectively, inmammalian expression vectors. Alternatively a human Ig expression vectorsuch as TCAE 5.3 is used that contains the human IgG1 heavy-chain andkappa light-chain constant region genes on the same plasmid (Preston etal., 1998, Infection & Immunity 66:4137-42). Expression vectors encodingchimeric heavy- and light-chains are then co-transfected into Chinesehamster ovary (CHO) cells for chimeric antibody productionImmunoreactivity and affinity of chimeric antibodies are compared toparental murine antibodies by ELISA and FACS.

Humanized Antibodies

As chimeric antibody therapeutics are still frequently antigenic,producing a human anti-chimeric antibody (HACA) immune response,chimeric antibodies against a non-ligand binding membrane proximaldomain of the extracellular domain of a Notch1 receptor can requirefurther humanization. To generate humanized antibodies the three shorthypervariable sequences, or complementary determining regions (CDRs), ofthe chimeric antibody heavy- and light-chain variable domains describedabove are engineered using recombinant DNA technology into the variabledomain framework of a human heavy- and light-chain sequences,respectively, and then cloned into a mammalian expression vector forexpression in CHO cells. The immunoreactivity and affinity of thehumanized antibodies are compared to parental chimeric antibodies byELISA and FACS. Additionally, site-directed or high-density mutagenesisof the variable region can be used to optimize specificity, affinity,etc. of the humanized antibody.

Example 2

Humanized antibodies against a membrane proximal region of theextracellular domain of a human Notch1 were generated. The variabledomains of the murine monoclonal antibody 52M51 were isolated andsequenced from the hybridoma line using degenerate PCR essentially asdescribed in Larrick, J. M., et al., 1989, Biochem. Biophys. Res. Comm.160: 1250 and Jones, S.T. & Bendig, M. M., 1991, Bio/Technology 9: 88.Human heavy and light chain variable framework regions likely to bestructurally similar to the parental 52M51 antibody amino acid sequencesare then considered as reference human framework regions to help guidethe design of novel synthetic frameworks. To identify the humanframework regions bearing similarity to 52M51 murine frameworks, thepredicted protein sequences encoded by the V_(H) and V_(L) murinevariable domains of 52M51 are compared with human antibody sequencesencoded by expressed human cDNA using BLAST searches for human sequencedeposited in Genbank. Using this method, expressed human cDNA sequences(e.g. genbank DA975021, DB242412) and germline Vh domains (e.g.IGHV1-24) were selected for further analysis in designing heavy chainframeworks. Similarly, expressed human cDNA sequences (e.g. genbankCD709370, CD707373) and germline Vl (e.g. IGLV7-46, IGLV8-61) wereconsidered in designing light chain frameworks.

The amino acid differences between candidate humanized framework heavychains and the parent murine monoclonal antibody 52M51 heavy chainvariable domain and light chain variable domains were evaluated forlikely importance, and a judgment made as to whether each difference inposition contributes to proper folding and function of the variabledomain. This analysis was guided by examination of solved crystalstructures of other antibody fragments (e.g., the structure of Fab 2E8as described in Trakhanov et al, Acta Crystallogr D Biol Crystallogr,1999, 55:122-28, as well as other protein crystal structures (e.g.,protein data bank structures 1ADQ and 1GIG)). Structures were modeledusing computer software including Jmol, quick PDB, and Pymol.Consideration was given to the potential impact of an amino acid at agiven position on the packing of the β-sheet framework, the interactionbetween the heavy and light chain variable domains, the degree ofsolvent exposure of the amino acid side chain, and the likelihood thatan amino acid would impact the positioning of the CDR loops. From thisanalysis, nine candidate V_(H) chains fused in-frame to the human IgG2constant region and eight candidate Vl chains fused in frame with thehuman IgLC1 constant region were conceived and chemically synthesized.The candidate heavy chains comprise: i) a synthetic framework designedto resemble natural human frameworks and ii) the parental 52M51 murineantibody CDRs.

The functionality of each candidate variant humanized heavy and lightchain was tested by cotransfection into mammalian cells. Each of thenine candidate humanized 52M51 heavy chains described above wascotransfected into HEK 293 cells with the murine 52M51 light chain cDNA,and conditioned media was assayed by ELISA for Notch1 binding activity.The 52M51 heavy chain variant exhibiting the most robust binding wasselected. This variant “52M51-H4” (SEQ ID NO:22) contains, in additionto murine CDRs, variation at 3 framework positions within the Vhframework, Kabat positions 20, 48, and 71 in comparison with an examplehuman framework (e.g. IGHV1-24). The 52M51-H4 humanized heavy chain wasthen cotransfected into HEK293 cells with each of the eight candidatehumanized light chains, and conditioned media was again assayed forantigen binding by ELISA. Two light chain variants “2M51L3” (SEQ IDNO:26) and “52M51L4” (SEQ ID NO:30) were found to exhibit better bindingthan the other candidates and were chosen for further study. Variant52M51-L3 contains, in addition to murine CDRs, variation at 1 frameworkposition at Kabat position 49 in comparison to an example humanframework (e.g., IGLV7-46). Two humanized variant antibodies, 52M51H4L3and 52M51H4L4, were developed. 52M51H4L3, as encoded by DNA depositedwith the ATCC, under the conditions of the Budapest Treaty on Oct. 15,2008, and assigned designation number PTA-9549.

The affinities for human and mouse Notch1 were determined using aBiacore 2000 instrument. Briefly, recombinant human and mouse Notch1proteins were immobilized on a CM5 chip using standard amine basedchemistry (NHS/EDC). Different antibody concentrations were injectedover the protein surfaces and kinetic data were collected over time. Thedata was fit using the simultaneous global fit equation to yielddissociation constants (K_(D), nM) for each Notch1 (Table 2).

TABLE 2 IgG Dissociation Constants (K_(D)) Antibody Human Notch1 (nM)Mouse Notch1 (nM) 52M51 2.86 NB 52M51H4L3 4.33 NB 52M51H4L4 7.35 NB

Example 3 Notch Receptor Signaling

In certain embodiments, the ability of Notch1 receptor antibodies toblock ligand-mediated Notch signaling was determined. In certainembodiments, HeLa cells engineered to overexpress Notch1 (Notch1-Hela)cultured in DMEM supplemented with antibiotics and 10% FCS wereco-transfected with 1) pGL4 8×CBS firefly luciferase containing a Notchresponsive promoter upstream of a firefly luciferase reporter gene tomeasure Notch signaling levels in response to DLL4 ligand; and 2) aRenilla luciferase reporter (Promega; Madison, Wis.) as an internalcontrol for transfection efficiency. Transfected cells were added tocultures plates coated overnight with 200 ng/well of hDLL4-fc protein,and antibodies to Notch1 were then added to the cell culture medium.Forty-eight hours following transfection, luciferase levels weremeasured using a dual luciferase assay kit (Promega; Madison, Wis.) withfirefly luciferase activity normalized to Renilla luciferase activity.The ability of antibodies to inhibit Notch1 pathway activation was thusdetermined Antibodies 52M51, 52M63, 52M74, and 52M80, generated againsta membrane proximal region of the extracellular domain of a human Notch1(FIG. 1A) significantly reduced luciferase activity indicative ofreduced Notch1 signaling as compared to other Notch1 antibodies (FIG.1B). Further, a humanized variant of antibody 52M51, variant 52M51 H4/L3displayed similar potency in reducing luciferase activity (FIG. 1C).

Notch Receptor Activation and ICD Formation

Cleavage of Notch receptors by furin, ADAM, and gamma-secretase resultsin formation of the Notch intracellular domain (ICD) that then triggersdownstream Notch signaling in the nucleus. In certain embodiments, theability of Notch1 receptor antibodies to block ligand-mediated receptoractivation was determined by Western blot analysis. Notch1-Hela cellswere grown in suspension culture in 293-SMII media (Gibco). Culturedcells were transferred to 96-well plates in which select wells had beenpre-coated with human DLL4-fc fusion protein (2 μg/ml) in DMEM plus 2%FBS and 1 μM MG132 (Calbiochem). Antibodies to generated against amembrane proximal region of the extracellular domain of human Notch1were added to the cell culture medium, and cells were incubated at 37°C. for five hours. Wells were then aspirated and the cells resuspendedin 2×SDS running buffer. Samples were sonicated at room temperature, andthen subjected to SDS-PAGE and western blot analysis using an antibodyspecific for the cleaved Notch1 ICD according to the manufacturer'srecommendations (Cell Signaling Technology). 52M51 along with 52M63,52M74, and 52M80 all significantly inhibited the generation of ICD afterligand stimulation (FIG. 1D).

Example 4 In Vivo Prevention of Tumor Growth Using Non-Ligand BindingRegion Anti-Notch Receptor Antibodies

Tumor cells from a patient sample that have been passaged as a xenograftin mice were prepared for injection into experimental animals. Tumorswere established at OncoMed Pharmaceuticals by adhering to proceduresdescribed previously (See Al-Hajj et al., 2003; Dalerba et al., 2007)and include: UM-PE13 and T3 (breast carcinoma cells), OMP-C9, OMP-C8,OMP-C6, and Colo-205 (colon tumor cells); and OMP-PN4 (pancreaticcarcinoma cells). Tumor tissue was removed under sterile conditions, cutup into small pieces, minced completely using sterile blades, and singlecell suspensions obtained by enzymatic digestion and mechanicaldisruption. The resulting tumor pieces were mixed with ultra-purecollagenase III in culture medium (200-250 units of collagenase per mL)and incubated at 37° C. for 3-4 hours with pipetting up and down througha 10-mL pipette every 15-20 min. Digested cells were filtered through a45 ul nylon mesh, washed with RPMI/20% FBS, and washed twice with HBSS.Dissociated tumor cells were then injected subcutaneously into NOD/SCIDmice at 6-8 weeks to elicit tumor growth. For UM-PE13 and T3 breasttumor cells, 50,000 cells in 100 ul were injected into the right mammaryfat pad (n=20) along with the implantation of an estrogen pellet. ForOMP-C9 colon tumor cells, 50,000 cells in 100 ul were injected into theright flank region (n=20). For OMP-C8 colon tumor cells, 10,000 cells in100 ul were injected into the right flank area (n=10). For OMP-C6 colontumor cells, 10,000 cells in 100 ul were injected into the right flank(n=10). All tumor cells were injected in a mixture of PBS (withoutmagnesium or calcium) and BD Matrigel (BD Biosciences) at a 1:1 ratio.

Three days after tumor cell injection, antibody treatment was commenced.Each injected animal received 10 mg/kg anti-Notch1 antibodies or PBS asa control intraperitoneal (i.p.) two times per week for a total of 6 to8 weeks Animals injected with PE13 cells received injections into theright upper mammary fat pad in addition to estrogen pellet injectionsAnimals injected with C9, C8, or C6 cells received injections in theright lower quadrant of the abdomen. Tumor size was assessed twice aweek.

In certain embodiments, antibodies against a membrane proximal region ofthe extracellular domain of human Notch1 were tested for an effect onthe formation of breast tumors. PE13 breast tumor cells (50,000 cellsper injection) were implanted subcutaneously into the mammary fat pads.Two days following cell implantation, animals were treated with eithercontrol antibody or 52M antibodies 52M1, 52M2, and 52M8 (which werewithout anti-Notch signaling capability, see FIG. 1B) at 10 mg/kg dosedi.p. twice a week. Treatment with non-Notch1 inhibitor antibodies had noeffect on tumor growth compared to control treated animals (FIGS. 2C and2D). In certain embodiments, animals injected with PE13 breast tumorcells are treated with either control antibody or 52M51 at 10 mg/kgdosed i.p. twice a week. Tumor volume is measured twice weekly, and theeffect of 52M51 on breast tumor growth is determined.

In alternative embodiments, dissociated tumor cells are first sortedinto tumorigenic and non-tumorigenic cells based on cell surface markersbefore injection into experimental animals. Specifically, tumor cellsdissociated as described above are washed twice with Hepes bufferedsaline solution (HBSS) containing 2% heat-inactivated calf serum (HICS)and resuspended at 10⁶ cells per 100 ul. Antibodies are added and thecells incubated for 20 min on ice followed by two washes with HBSS/2%HICS. Antibodies include anti-ESA (Biomeda, Foster City, Calif.),anti-CD44, anti-CD24, and Lineage markers anti-CD2, -CD3, -CD10, -CD16,-CD18, -CD31, -CD64, and -CD140b (collectively referred to as Lin;PharMingen, San Jose, Calif.). Antibodies are directly conjugated tofluorochromes to positively or negatively select cells expressing thesemarkers. Mouse cells are eliminated by selecting against H2 Kd+ cells,and dead cells are eliminated by using the viability dye 7AAD. Flowcytometry is performed on a FACSVantage (Becton Dickinson, FranklinLakes, N.J.). Side scatter and forward scatter profiles are used toeliminate cell clumps. Isolated ESA+, CD44+, CD24−/low, Lin− tumorigeniccells are then injected subcutaneously into NOD/SCID mice to elicittumor growth.

Example 5 In Vivo Treatment of Tumors Using Anti-Notch1 ReceptorAntibodies

Tumor cells from a patient sample (solid tumor biopsy or pleuraleffusion) that have been passaged as a xenograft in mice were preparedfor repassaging into experimental animals. Tumor tissue was removed, cutup into small pieces, minced completely using sterile blades, and singlecell suspensions obtained by enzymatic digestion and mechanicaldisruption. Dissociated tumor cells were then injected subcutaneouslyinto the mammary fat pads, for breast tumors, or into the flank, fornon-breast tumors, of NOD/SCID mice to elicit tumor growth. In certainembodiments, ESA+, CD44+, CD24−/low, Lin-tumorigenic tumor cells areisolated as described in detail above and injected.

In certain embodiments, freshly isolated C8 colon tumor cells (225 cellsper animal) were implanted subcutaneously into NOD/SCID mice. Followingtumor cell injection, animals were monitored for tumor growth. Tumorswere allowed to grow for 48 days until they reached an average size ofapproximately 210 mm³ and randomized into two groups (n=10 per group).The animals were treated with either control antibody or antibody thatbinds to the membrane proximal region of the extracellular domain ofhuman Notch1, 52M51, (10 mg/kg) dosed i.p. twice a week. Tumor size wasassessed on days 55, 57, and 62. Animals treated with 52M51 showed astatistically significant (p=0.0006) inhibition of tumor growth comparedto control treated animals (FIGS. 2A and 2B).

At the end point of antibody treatment, tumors are harvested for furtheranalysis. In some embodiments, a portion of the tumor is analyzed byimmunofluorescence to assess antibody penetration into the tumor andtumor response. A portion of each harvested tumor from anti-Notch1receptor treated and control antibody treated mice is flash-frozen inliquid nitrogen, embedded in O.C.T., and cut on a cryostat as 10 umsections onto glass slides. Alternatively a portion of each tumor isformalin-fixed, paraffin-embedded, and cut on a microtome as 10 umsection onto glass slides. Sections are post-fixed and incubated withchromophore labeled antibodies that specifically recognize injectedantibodies to detect anti-NOTCH1 receptor or control antibodies presentin the tumor biopsy. Furthermore antibodies that detect different tumorand tumor recruited cell types such as, for example, anti-VE cadherin(CD 144) or anti-PECAM-1 (CD31) antibodies to detect vascularendothelial cells, anti-smooth muscle alpha-actin antibodies detectvascular smooth muscle cells, anti-Ki67 antibodies to detectproliferating cells, TUNEL assays to detect dying cells, andanti-intracellular domain (ICD) Notch fragment antibodies to detectNotch signaling can be used to assess affects of antibody treatment onangiogenesis, tumor growth and tumor morphology.

The effect of anti-Notch1 receptor antibody treatment on tumor cell geneexpression is also assessed. Total RNA is extracted from a portion ofeach harvested tumor from Notch1 antibody treated and control antibodytreated mice and used for quantitative RT-PCR. Expression levels ofNotch1, components of Notch signaling pathway including, as well asaddition cancer stem cell markers previously identified including, forexample, CD44 are analyzed relative to the house-keeping gene GAPDH asan internal control. Changes in tumor cell gene expression upon Notch1receptor antibody treatment are thus determined.

In addition, the effect of anti-Notch1 receptor antibody treatment onthe presence of cancer stem cells in a tumor is assessed. Tumor samplesfrom Notch1 versus control antibody treated mice are cut up into smallpieces, minced completely using sterile blades, and single cellsuspensions obtained by enzymatic digestion and mechanical disruption.Dissociated tumor cells are then analyzed by FACS analysis for thepresence of tumorigenic cancer stem cells based on ESA+, CD44+,CD24−/low, Lin-surface cell marker expression as described in detailabove.

The tumorigenicity of cells isolated based on ESA+, CD44+, CD24−/low,Lin− expression following anti-Notch1 antibody treatment can then beassessed. 5,000, 1,000, 500, and 100 isolated ESA+, CD44+, CD24−/low,Lin− cancer stem cells from Notch1 antibody treated versus controlantibody treated mice are re-injected subcutaneously into the mammaryfat pads of NOD/SCID mice. The tumorigenicity of cancer stem cells basedon the number of injected cells required for consistent tumor formationis thus determined.

In contrast to the in vivo efficacy of 52M51, an antibody that inhibitsNotch1 signaling, in a colon xenograft model described above, certainother antibodies that recognize the membrane proximal region of Notch 1,but don't inhibit Notch 1 signaling, were found to not have anti-tumorefficacy in vivo in a breast xenograft model. The antibodies 52M51,52M2, and 52M8, each of which had been found to not appreciably inhibitNotch signaling (Example 3 and FIG. 1B), were injected in NOD/SCID micewhich had been previously injected with PE13 breast tumor cells. Each ofthe antibodies 52M1, 52M2, and 52M8 failed to effect tumor growth in thexenograft model when compared against control-treated animals (FIG. 2C(52M1, 52M2) and FIG. 2D (52M8)).

Example 6 Treatment of Human Cancer Using Anti-Notch Receptor Antibodies

This example describes methods for treating cancer using antibodiesagainst a Notch receptor to target tumors comprising cancer stem cellsand/or tumor cells in which Notch receptor expression has been detected.

The presence of cancer stem cell marker expression can first bedetermined from a tumor biopsy. Tumor cells from a biopsy from a patientdiagnosed with cancer are removed under sterile conditions. In someembodiments, the tissue biopsy is fresh-frozen in liquid nitrogen,embedded in O.C.T., and cut on a cryostat as 10 um sections onto glassslides. Alternatively the tissue biopsy is formalin-fixed,paraffin-embedded, and cut on a microtome as 10 um section onto glassslides. Sections are incubated with antibodies against a Notch receptorto detect protein expression. Additionally, the presence of cancer stemcells can be determined Tissue biopsy samples are cut up into smallpieces, minced completely using sterile blades, and cells subject toenzymatic digestion and mechanical disruption to obtain a single cellsuspension. Dissociated tumor cells are then incubated with anti-ESA,-CD44, -CD24, -Lin, and -Notch1 antibodies to detect cancer stem cells,and the presence of ESA+, CD44+, CD24−/low, Lin-, Notch+ tumor stemcells is determined by flow cytometry as described in detail above.

Cancer patients whose tumors are diagnosed as expressing a Notchreceptor are treated with anti-Notch receptor antibodies. Humanized orhuman monoclonal anti-Notch receptor antibodies generated as describedabove are purified and formulated with a suitable pharmaceutical carrierin PBS for injection. Patients are treated with the Notch antibodiesonce a week for at least 10 weeks, but in certain cases once a week forat least about 14 weeks. Each administration of the antibody should be apharmaceutically effective dose about 2 to about 100 mg/ml and incertain cases between about 5 to about 40 mg/ml. The antibody can beadministered prior to, concurrently with, or after standard radiotherapyregimens or chemotherapy regimens using one or more chemotherapeuticagent, such as oxaliplatin, fluorouracil, leucovorin, or streptozocin.Patients are monitored to determine whether such treatment has resultedin an anti-tumor response, for example, based on tumor regression,reduction in the incidences of new tumors, lower tumor antigenexpression, decreased numbers of cancer stem cells, or other means ofevaluating disease prognosis.

Example 7 Additional Studies of In Vivo Treatment of Tumors UsingAnti-Notch1 Receptor Antibodies

In one embodiment, M2 melanoma cells (10,000) were injectedsubcutaneously in NOD-SCID mice. Tumors were allowed to grow for 35 daysuntil they had reached a volume of approximately 110 mm³ Tumor-bearingmice were randomized into two groups (n=10) and treated with eithercontrol antibody or anti-Notch1 antibody 52R43. Antibodies were dosedtwice weekly at 10 mg/kg. Tumor volumes were measured on the indicateddays. As shown in FIG. 3A, anti-Notch1 treatment with 52R43 reducedtumor growth relative to the control group (p=0.02).

In one embodiment, Lu24 lung tumor cells (30,000) were injectedsubcutaneously in NOD-SCID mice. Tumors were allowed to grow for 35 daysuntil they had reached a volume of approximately 205 mm³ Tumor-bearingmice were randomized into two groups (n=8) and treated with eithercontrol antibody or anti-Notch1 antibody 52R43. Antibodies were dosedtwice weekly at 10 mg/kg. Tumor volumes were measured on the indicateddays. As shown in FIG. 3B, anti-Notch1 treatment with 52R43 reducedtumor growth relative to the control group (p=0.04).

In one embodiment, PN8 pancreatic tumor cells (50,000) were injectedsubcutaneously in NOD-SCID mice. Tumors were allowed to grow for 27 daysuntil they had reached a volume of approximately 115 mm³ Tumor bearingmice were randomized into two groups (n=8) and treated with eithercontrol antibody or anti-Notch1 antibody 52R43. Antibodies were dosedtwice weekly at 10 mg/kg. Tumor volumes were measured on the indicateddays. As shown in FIG. 3C, anti-Notch1 treatment with 52R43 reducedtumor growth relative to the control group (p=0.005).

In one embodiment, T1 breast tumor cells (300,000) were injectedsubcutaneously in NOD-SCID mice. Tumors were allowed to grow for 27 daysuntil they had reached a volume of approximately 130 mm³ Tumor bearingmice were randomized into four groups (n=10) and treated with eithercontrol antibody, anti-Notch1 52R43, taxol, or a combination of 52R43and taxol. Antibodies were dosed once weekly at 15 mg/kg and taxol wasdosed once weekly at 12 mg/kg. Tumor volumes were measured on theindicated days. As shown in FIG. 3D, anti-Notch1 treatment with 52R43reduced tumor growth relative to the control group (p<0.0001), and thecombination group was reduced relative to taxol alone (p=0.001)

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those in the relevant fields areintended to be within the scope of the following claims.

SEQUENCES Notch1 Polynucleotide encoding amino acids 1427-1732.SEQ ID NO: 1CACATCCTGGACTACAGCTTCGGGGGTGGGGCCGGGCGCGACATCCCCCCGCCGCTGATCGAGGAGGCGTGCGAGCTGCCCGAGTGCCAGGAGGACGCGGGCAACAAGGTCTGCAGCCTGCAGTGCAACAACCACGCGTGCGGCTGGGACGGCGGTGACTGCTCCCTCAACTTCAATGACCCCTGGAAGAACTGCACGCAGTCTCTGCAGTGCTGGAAGTACTTCAGTGACGGCCACTGTGACAGCCAGTGCAACTCAGCCGGCTGCCTCTTCGACGGCTTTGACTGCCAGCGTGCGGAAGGCCAGTGCAACCCCCTGTACGACCAGTACTGCAAGGACCACTTCAGCGACGGGCACTGCGACCAGGGCTGCAACAGCGCGGAGTGCGAGTGGGACGGGCTGGACTGTGCGGAGCATGTACCCGAGAGGCTGGCGGCCGGCACGCTGGTGGTGGTGGTGCTGATGCCGCCGGAGCAGCTGCGCAACAGCTCCTTCCACTTCCTGCGGGAGCTCAGCCGCGTGCTGCACACCAACGTGGTCTTCAAGCGTGACGCACACGGCCAGCAGATGATCTTCCCCTACTACGGCCGCGAGGAGGAGCTGCGCAAGCACCCCATCAAGCGTGCCGCCGAGGGCTGGGCCGCACCTGACGCCCTGCTGGGCCAGGTGAAGGCCTCGCTGCTCCCTGGTGGCAGCGAGGGTGGGCGGCGGCGGAGGGAGCTGGACCCCATGGACGTCCGCGGCTCCATCGTCTACCTGGAGATTGACAACCGGCAGTGTGTGCAGGCCTCCTCGCAGTGCTTCCAGAGTGCCACCGACGTGGCCGCATTCCTGGGAGCGCTCGCCTCGCTGGGCAGCCTCAACATCCCCTACAAGATCGAGGCCGTGCAGAGTGAGACCGTGGAGCCGCCCCCGCCG Notch1 amino acids 1427-1732 SEQ ID NO: 2HILDYSFGGGAGRDIPPPLIEEACELPECQEDAGNKVCSLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQRAEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVVVVLMPPEQLRNSSFHFLRELSRVLHTNVVFKRDAHGQQMIFPYYGREEELRKHPIKRAAEGWAAPDALLGQVKASLLPGGSEGGRRRRELDPMDVRGSIVYLEIDNRQCVQASSQCFQSATDVAAFLGALASLGSLNIPYKIEAVQSET VEPPPPMouse antibody 52M51 sequences:52M51 Light chain polynucleotide sequence (Putative signalsequence is underlined) SEQ ID NO: 3ATGGCCTGGATTTCACTTATACTCTCTCTCCTGGCTCTCAGCTCAGGGGCCATTTCCCAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTACGCCAACTGGGTCCAAGAAAAACCTGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCACTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCCACCTTCCTCTGAAGAGCTCGAGACTAACAAGGCCACACTGGTGTGTACGATCACTGATTTCTACCCAGGTGTGGTGACAGTGGACTGGAAGGTAGATGGTACCCCTGTCACTCAGGGTATGGAGACAACCCAGCCTTCCAAACAGAGCAACAACAAGTACATGGCTAGCAGCTACCTGACCCTGACAGCAAGAGCATGGGAAAGGCATAGCAGTTACAGCTGCCAGGTCACTCATGAAGGTCACACTGTGGAGAAGAGTTTGTCCCGTGCTGACTGTTCCTAG52M51 Light chain amino acid sequence (Putative signal sequenceis underlined) SEQ ID NO: 4MAWISLILSLLALSSGAISQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS52M51 Light chain variable region polynucleotide sequence(Putative signal sequence is underlined) SEQ ID NO: 5ATGGCCTGGATTTCACTTATACTCTCTCTCCTGGCTCTCAGCTCAGGGGCCATTTCCCAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTACGCCAACTGGGTCCAAGAAAAACCTGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCACTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGC52M51 Light chain variable region amino acid sequence (Putativesignal sequence is underlined) SEQ ID NO: 6MAWISLILSLLALSSGAISQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQG52M51 Light chain variable region polynucleotide sequencewithout putative signal sequence SEQ ID NO: 7CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTACGCCAACTGGGTCCAAGAAAAACCTGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCACTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGC52M51 Light chain variable region amino acid sequence withoutputative signal sequence SEQ ID NO: 8QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLG52M51 Heavy chain polynucleotide sequence (Putative signalsequence is underlined) SEQ ID NO: 9ATGGAATGGACCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGCAGGTGTCCACTCCCAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTGCTGGCTACACAATGAGAGGCTACTGGATAGAGTGGATAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGACAGATTTTACCTGGAACTGGGAGAACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCAACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGATTTGATGGTAACTACGGTTACTATGCTATGGACTACTGGGGTCAAGGATCCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCCCTCGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTCCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATATCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATAACAGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGAACACGAATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA52M51 Heavy chain amino acid sequence (Putative signal sequenceis underlined) SEQ ID NO: 10MEWTWVFLFLLSVTAGVHSQVQLQQSGAELMKPGASVKISCKAAGYTMRGYWIEWIKQRPGHGLEWIGQILPGTGRTNYNEKFKGKATFTADTSSNTANMQLSSLTSEDSAVYYCARFDGNYGYYAMDYWGQGSSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK52M51 Heavy chain variable region polynucleotide sequence(Putative signal sequence is underlined) SEQ ID NO: 11ATGGAATGGACCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGCAGGTGTCCACTCCCAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTGCTGGCTACACAATGAGAGGCTACTGGATAGAGTGGATAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGACAGATTTTACCTGGAACTGGGAGAACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCAACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGATTTGATGGTAACTACGGTTACTATGCTATGGACTACTGGGGTCAAGGATCCTCAGTCACCGTCTCCTCA52M51 Heavy chain variable region amino acid sequence (Putativesignal sequence is underlined) SEQ ID NO: 12MEWTWVFLFLLSVTAGVHSQVQLQQSGAELMKPGASVKISCKAAGYTMRGYWIEWIKQRPGHGLEWIGQILPGTGRTNYNEKFKGKATFTADTSSNTANMQLSSLTSEDSAVYYCARFDGNYGYYAMDYWGQGSSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVT52M51 Heavy chain variable region polynucleotide sequencewithout putative signal sequence SEQ ID NO: 13CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTGCTGGCTACACAATGAGAGGCTACTGGATAGAGTGGATAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGACAGATTTTACCTGGAACTGGGAGAACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCAACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGATTTGATGGTAACTACGGTTACTATGCTATGGACTACTGGGGTCAAGGATCCTCAGTCACCGTCTCC TCA52M51 Heavy chain variable region amino acid sequence withoutputative signal sequence SEQ ID NO: 14QVQLQQSGAELMKPGASVKISCKAAGYTMRGYWIEWIKQRPGHGLEWIGQILPGTGRTNYNEKFKGKATFTADTSSNTANMQLSSLTSEDSAVYYCARFDGNYGYYAMDYWGQGSSVTVS SA52M51 Heavy chain CDR1 SEQ ID NO: 15 RGYWIE 52M51 Heavy chain CDR2SEQ ID NO: 16 QILPGTGRTNYNEKFKG 52M51 Heavy chain CDR3 SEQ ID NO: 17FDGNYGYYAMDY 52M51 Light chain CDR1 SEQ ID NO: 18 RSSTGAVTTSNYAN52M51 Light chain CDR2 SEQ ID NO: 19 GTNNRAP 52M51 Light chain CDR3SEQ ID NO: 20 ALWYSNHWVFGGGTKL Humanized 52M51 sequences:52M51-H4 Heavy chain polynucleotide sequence (Putative signalsequence underlined) SEQ ID NO: 21ATGGATTGGACATGGAGGGTGTTCTGCCTCCTCGCTGTGGCTCCTGGAGTCCTGAGCCAGGTCCAGCTCGTCCAGAGCGGGGCTGAAGTCAAGAAGCCTGGCGCTAGCGTCAAAATCAGCTGTAAGGTCAGCGGATACACACTGAGGGGATACTGGATCGAGTGGGTGAGGCAGGCTCCAGGAAAGGGCCTGGAATGGATCGGCCAGATCCTGCCTGGAACCGGAAGGACAAATTACAATGAGAAGTTTAAGGGAAGGGTCACAATGACAGCAGACACAAGCACAGACACAGCTTATATGGAACTCAGCTCCCTCAGATCCGAGGACACCGCTGTCTACTATTGTGCCAGGTTCGATGGAAATTACGGATACTATGCCATGGATTACTGGGGACAGGGGACAACGGTCACCGTGAGCTCAGCCAGCACAAAGGGCCCTAGCGTCTTCCCTCTGGCTCCCTGCAGCAGGAGCACCAGCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA52M51 H4 Heavy chain amino acid sequence (Putative signalsequence underlined) SEQ ID NO: 22MDWTWRVFCLLAVAPGVLSQVQLVQSGAEVKKPGASVKISCKVSGYTLRGYWIEWVRQAPGKGLEWIGQILPGTGRTNYNEKFKGRVTMTADTSTDTAYMELSSLRSEDTAVYYCARFDGNYGYYAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK52M51-H4 Heavy chain variable region amino acid sequence(Putative signal sequence underlined) SEQ ID NO: 23MDWTWRVFCLLAVAPGVLSQVQLVQSGAEVKKPGASVKISCKVSGYTLRGYWIEWVRQAPGKGLEWIGQILPGTGRTNYNEKFKGRVTMTADTSTDTAYMELSSLRSEDTAVYYCARFDGNYGYYAMDYWGQGTTVTVSSA52M51-H4 Heavy chain variable region amino acid sequencewithout putative signal sequence SEQ ID NO: 24QVQLVQSGAEVKKPGASVKISCKVSGYTLRGYWIEWVRQAPGKGLEWIGQILPGTGRTNYNEKFKGRVTMTADTSTDTAYMELSSLRSEDTAVYYCARFDGNYGYYAMDYWGQGTTVTVS SA52M51-L3 Light chain polynucleotide sequence (Putative signalsequence is underlined) SEQ ID NO: 25ATGAGCGTCCCTACAATGGCTTGGATGATGCTCCTGCTGGGACTCCTGGCTTATGGAAGCGGAGTGGATAGCCAGGCCGTCGTCACACAGGAACCTAGCCTCACCGTTAGCCCTGGAGGAACAGTCACACTGACCTGTAGGAGCTCCACAGGAGCTGTGACAACAAGCAATTACGCTAACTGGTTCCAGCAGAAGCCCGGTCAAGCCCCTAGAACCCTCATCGGCGGCACCAATAACAGAGCTCCCGGAGTCCCCGCCAGGTTCTCCGGCTCCCTCCTGGGTGGCAAGGCTGCTCTGACACTCAGCGGTGCCCAGCCAGAGGATGAAGCGGAGTACTACTGTGCACTGTGGTACAGCAACCATTGGGTTTTCGGAGGCGGAACAAAGTTAACCGTCCTCGGGCAGCCTAAGGCTGCTCCTAGCGTCACACTGTTCCCCCCATCTAGCGAGGAGCTGCAGGCTAACAAGGCAACCCTCGTCTGCCTGGTTAGCGACTTCTACCCTGGCGCTGTCACAGTGGCCTGGAAAGCTGACGGCTCCCCTGTGAAAGTTGGCGTCGAAACCACAAAGCCTTCTAAGCAGAGCAATAATAAATATGCCGCAAGCTCCTACCTCTCCCTGACTCCTGAGCAGTGGAAAAGCCATAGGAGCTACTCCTGCCGGGTCACACACGAAGGAAGCACAGTGGAAAAGACAGTCGCCCCTGCTGAGTGTAGCTGA52M51-L3 Light chain amino acid sequence (Putative signalsequence is underlined) SEQ ID NO: 26MSVPTMAWMMLLLGLLAYGSGVDSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRTLIGGTNNRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS52M51-L3 Light chain variable region amino acid sequence(Putative signal sequence is underlined) SEQ ID NO: 27MSVPTMAWMMLLLGLLAYGSGVDSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRTLIGGTNNRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVLG 52M51-L3 Light chain variable region amino acid sequencewithout putative signal sequence SEQ ID NO: 28SGVDSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRTLIGGTNNRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVLG52M51-L4 Light chain polynucleotide sequence (Putative signalsequence is underlined) SEQ ID NO: 29ATGAGCGTCCCTACAATGGCTTGGATGATGCTCCTGCTGGGACTCCTGGCTTATGGAAGCGGAGTGGATAGCCAGACCGTCGTCACACAGGAACCTAGCTTTTCCGTTAGCCCTGGAGGAACAGTCACACTGACCTGTAGGAGCTCCACAGGAGCTGTGACAACAAGCAATTACGCTAACTGGTATCAGCAGACTCCCGGTCAAGCCCCTAGAACCCTCATCGGCGGCACCAATAACAGAGCTCCCGGAGTCCCCGACAGGTTCTCCGGCTCCATCCTGGGAAATAAAGCTGCTCTGACAATCACAGGTGCCCAGGCTGACGATGAAAGCGACTACTACTGTGCACTGTGGTACAGCAACCATTGGGTTTTCGGAGGCGGAACAAAGTTAACCGTCCTCGGGCAGCCTAAGGCTGCTCCTAGCGTCACACTGTTCCCCCCATCTAGCGAGGAGCTGCAGGCTAACAAGGCAACCCTCGTCTGCCTGGTTAGCGACTTCTACCCTGGCGCTGTCACAGTGGCCTGGAAAGCTGACGGCTCCCCTGTGAAAGTTGGCGTCGAAACCACAAAGCCTTCTAAGCAGAGCAATAATAAATATGCCGCAAGCTCCTACCTCTCCCTGACTCCTGAGCAGTGGAAAAGCCATAGGAGCTACTCCTGCCGGGTCACACACGAAGGAAGCACAGTGGAAAAGACAGTCGCCCCTGCTGAGTGTAGCTGA52M51-L4 Light chain amino acid sequence (Putative signalsequence is underlined) SEQ ID NO: 30MSVPTMAWMMLLLGLLAYGSGVDSQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWYQQTPGQAPRTLIGGTNNRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNHWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS52M51-L4 Light chain variable region amino acid sequence(Putative signal sequence is underlined) SEQ ID NO: 31MSVPTMAWMMLLLGLLAYGSGVDSQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWYQQTPGQAPRTLIGGTNNRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNHWVFGGGTKLTVLG 52M51-L4 Light chain variable region amino acid sequencewithout putative signal sequence SEQ ID NO: 32SGVDSQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWYQQTPGQAPRTLIGGTNNRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNHWVFGGGTKLTVLG

1-84. (canceled)
 85. An isolated polynucleotide encoding an antibodythat specifically birds human NOTCH1, wherein the antibody comprises:(a) a heavy chain CDR1 comprising RGYWIE (SEQ ID NO:15); a heavy chainCDR2 comprising QILPGTGRTNYNEKFKG (SEQ ID NO:16); and a heavy chain CDR3comprising FDGNYGYYAMDY (SEQ ID NO:17); and (b) a light chain CDR1comprising RSSTGAVTTSNYAN (SEQ ID NO:18); a light chain CDR2 comprisingGTNNRAP (SEQ ID NO:19); and a light chain CDR3 comprisingALWYSNHWVFGGGTKL (SEQ ID NO:20).
 86. The isolated polynucleotide ofclaim 85, wherein the antibody is a monoclonal antibody, a chimericantibody, a humanized antibody, a human antibody, or an antibodyfragment.
 87. A vector comprising the polynucleotide of claim
 85. 88. Anisolated cell comprising the polynucleotide of claim
 85. 89. An isolatedcell comprising the vector of claim
 87. 90. An isolated polynucleotideencoding an antibody that specifically binds human NOTCH1, wherein theantibody comprises: (a) a heavy chain variable region having at least90% sequence identity to SEQ ID NO:14 or SEQ ID NO:24; and/or (b) alight chain variable region having at least 90% sequence identity to SEQID NO:8, SEQ ID NO:28, or SEQ ID NO:32.
 91. The polynucleotide of claim90, wherein the antibody comprises: (a) a heavy chain variable regionhaving at least about 90% sequence identity to SEQ ID NO:24; and (b) alight chain variable region having at least about 90% sequence identityto SEQ ID NO:28.
 92. The polynucleotide of claim 91, wherein theantibody comprises: (a) a heavy chain variable region comprising SEQ IDNO:24; and (b) a light chain variable region comprising SEQ ID NO:28.93. The polynucleotide of claim 91, wherein the antibody comprises: (a)a heavy chain variable region comprising SEQ ID NO:24; and (b) a lightchain variable region comprising SEQ ID NO:32.
 94. The polynucleotide ofclaim 90, wherein the antibody comprises: (a) a heavy chain variableregion having at least about 90% sequence identity to SEQ ID NO:14; and(b) a light chain variable region having at least about 90% sequenceidentity to SEQ ID NO:8.
 95. The polynucleotide of claim 94, wherein theantibody comprises: (a) a heavy chain variable region comprising SEQ IDNO:14; and (b) a light chain variable region comprising SEQ ID NO:8. 96.The isolated polynucleotide of claim 90, wherein the antibody is amonoclonal antibody, a chimeric antibody, a humanized antibody, a humanantibody, or an antibody fragment.
 97. A vector comprising thepolynucleotide of claim
 90. 98. An isolated cell comprising thepolynucleotide of claim
 90. 99. An isolated cell comprising the vectorof claim
 97. 100. An isolated polynucleotide encoding a polypeptidecomprising a sequence selected from the group consisting of SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:30, SEQ′ID NO:31, SEQ ID NO:32, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14. 101.The isolated polynucleotide of claim 100, wherein the polynucleotideencodes a polypeptide comprising the sequence of SEQ ID NO:24 and/or SEQTD NO:28.
 102. The isolated polynucleotide of claim 100, wherein thepolynucleotide encodes a polypeptide comprising the sequence of SEQ IDNO:22 and/or SEQ ID NO:26.
 103. An isolated polynucleotide comprising asequence selected from the group consisting of SEQ ID NO:21, SEQ IDNO:25, SEQ ID NO:29, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:11, and SEQ ID NO:13.
 104. The isolated polynucleotide ofclaim 103, comprising the sequence of SEQ ID NO:21 and/or SEQ ID NO:25.105. An isolated polynucleotide deposited with ATCC as PTA-9549.
 106. Anisolated polynucleotide that encodes an antibody produced by thehybridoma cell line deposited with ATCC as PTA-9405.